Target tissue-specific antigen-binding molecule

ABSTRACT

The present inventors discovered that the above-mentioned problems can be solved by producing antigen-binding molecules that contain an antigen-binding domain whose antigen-binding activity varies depending on the concentration of a target tissue-specific compound. Use of antigen-binding molecules of the present invention enables various diseases that originate from a target tissue to be treated in a manner specific to the target tissue.

TECHNICAL FIELD

The present invention provides antigen-binding molecules comprising anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a target tissue-specific compound; productionmethods and screening methods for the antigen-binding molecules; andpharmaceutical compositions containing the antigen-binding molecules.

BACKGROUND ART

Antibodies are drawing attention as pharmaceuticals as they are highlystable in plasma and have few side effects. In particular, a number ofIgG-type antibody pharmaceuticals are available on the market, and manyantibody pharmaceuticals are currently under development (Non-PatentDocuments 1 and 2).

As cancer therapeutic agents using antibody pharmaceuticals, Rituxanagainst a CD20 antigen, cetuximab against an EGFR antigen, herceptinagainst a HER2 antigen, and such have been approved so far (Non-PatentDocument 3). These antibody molecules bind to antigens expressed oncancer cells, and exhibit cytotoxic activity against cancer cells byADCC and such.

Such cytotoxic activity by ADCC and etc. are known to depend on thenumber of antigens expressed on cells targeted by the therapeuticantibodies (Non-Patent Document 4); therefore, high expression level ofthe target antigen is preferable from the stand point of the effects ofthe therapeutic antibodies. However, even if the antigen expressionlevel is high, when antigens are expressed in normal tissues, cytotoxicactivity mediated by ADCC etc will be exerted against normal cells, andtherefore side-effects will become a major problem. Therefore, antigenstargeted by therapeutic antibodies used as therapeutic agents for cancerare preferably antigens specifically expressed in cancer cells. Forexample, antibody molecules against the EpCAM antigen which is known asa cancer antigen have been considered to be promising as therapeuticagents for cancer. However, the EpCAM antigen is known to be expressedin the pancreas as well, and in practice, administration of anti-EpCAMantibodies in clinical trials has been reported to cause pancreatitis asa side-effect due to cytotoxic activity towards the pancreas (Non-PatentDocument 5).

Following the success of antibody pharmaceuticals that exert cytotoxicactivity by ADCC activity, a second generation of improved antibodymolecules that exert strong cytotoxic activity through enhancement ofADCC activity by removing fucose of N-type sugar chains in the nativehuman IgG1 Fc region (Non-Patent Document 6), enhancement of ADCCactivity by enhancing the binding toward FcγRIIIa by substitution ofamino acids in the native human IgG1 Fc region (Non-Patent Document 7),and such have been reported. As antibody pharmaceuticals that exertcytotoxic activity against cancer cells through a mechanism other thanthe above-mentioned ADCC activity mediated by NK cells, improvedantibody molecules that exert a stronger cytotoxic activity, such as anantibody-drug conjugate (ADC) in which an antibody is conjugated with adrug having potent cytotoxic activity (Non-Patent Document 8), and a lowmolecular weight antibody that exerts toxic activity against cancercells by recruiting T cells to cancer cells, have been reported as well.

Such antibody molecules exerting a stronger cytotoxic activity can exertcytotoxic activity against cancer cells that do not have much antigenexpression, but on the other hand, they will exert similar cytotoxicactivity against normal tissues with low antigen expression. In fact, incomparison to cetuximab which is a natural human IgG1 against an EGFRantigen, EGFR-BiTE, which is a bispecific antibody against CD3 and EGFR,can exert a potent cytotoxic activity against cancer cells by recruitingT cells to cancer cells and exert antitumor effects. On the other hand,since EGFR is expressed also in normal tissues, when EGFR-BiTE isadministered to cynomolgus monkeys, serious side effects have appeared(Non-Patent Document 10). Furthermore, bivatuzumab mertansine, an ADCformed by linking mertansine to an antibody against CD44v6 which ishighly expressed in cancer cells, has been shown to cause severe skintoxicity and liver toxicity in clinical practice because CD44v6 isexpressed also in normal tissues (Non-Patent Document 11).

When antibodies that can exert a potent cytotoxic activity againstcancer cells having low antigen expression are used as such, the targetantigen needs to be expressed in a highly cancer-specific manner.However, since HER2 and EGFR, which are target antigens of herceptin andcetuximab, respectively, are also expressed in normal tissues, thenumber of cancer antigens expressed in a highly cancer-specific manneris thought to be limited. Therefore, while it is possible to strengthenthe cytotoxic activity against cancer, the side effects occurring due tocytotoxic actions against normal tissues may become problematic.

Furthermore, recently, ipilimumab which enhances tumor immunity byinhibiting CTLA4 which contributes to immunosuppression in cancer wasshown to prolong overall survival of metastatic melanoma (Non-PatentDocument 12). However, since ipulimumab inhibits CTLA4 systemically,while tumor immunity is enhanced, the emergence of autoimmunedisease-like severe side effects due to systemic activation of theimmune system is becoming a problem (Non-Patent Document 13).

On the other hand, as antibody pharmaceuticals against diseases besidescancer, antibody pharmaceuticals that exert therapeutic effects byinhibiting inflammatory cytokines in inflammatory/autoimmune diseasesare known (Non-Patent Document 14). For example, Remicade and Humirawhich target TNF, and Actemra which targets IL-6R exhibit hightherapeutic effects against rheumatoid arthritis, but on the other hand,systemic neutralization of these cytokines has led to the observation ofinfection as side effects (Non-Patent Document 15).

Various techniques have been developed as techniques that can be appliedto second-generation antibody pharmaceuticals. While techniques forimproving effector functions, antigen-binding ability, pharmacokinetics,and stability, or techniques for reducing immunogenic risks have beenreported (Non-Patent Document 16), there are hardly any reports ontechniques that enable target tissue-specific action of antibodypharmaceuticals to overcome such side effects. For example, regardinglesions such as cancer tissues and inflammatory tissues, pH-dependentantibodies that make use of the acidic pH condition at these targettissues have been reported (Patent Documents 1 and 2). However, thedecrease of pH (that is, increase in hydrogen ion concentration) incancer tissues and inflammatory tissues as compared to normal tissues isslight, and since it is difficult to produce antibodies that act bydetecting a slight increase in the concentration of hydrogen ions whichhave an extremely small molecular weight, and also because acidic pHconditions may be found in normal tissues such as osteoclastic boneresorption region or in tissues other than the lesion of interest, useof pH conditions as a lesion-specific environmental factor wasconsidered to face many challenges. On the other hand, methods forproducing antibodies that exert antigen-binding activity only after theyare cleaved by a protease expressed at lesion sites such as cancertissues and inflammatory tissues have been reported (Patent Document 3).However, since cleavage of antibodies by proteases is irreversible, whenthe antibodies that have been cleaved at the lesion site enter the bloodstream and return to normal tissues, they can bind to the antigens innormal tissues as well, and this is considered to be a problem.Furthermore, cancer specificity of such proteases is also thought tohave problems that need to be addressed. Therefore, techniques thatenable reversible action at sites of inflammation or cancer (lesionsites) without systemic action in normal tissues and blood for exertingdrug efficacy while avoiding side effects are not known.

PRIOR ART DOCUMENTS Patent Documents

-   [Patent document 1] WO 2003/105757-   [Patent document 2] WO 2012/033953-   [Patent document 3] WO 2010/081173

Non-Patent Documents

-   [Non-patent document 1] Monoclonal antibody successes in the clinic.    Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C    Dewitz, Nat. Biotechnol. (2005) 23, 1073-1078-   [Non-patent document 2] The therapeutic antibodies market to 2008.    Pavlou A K, Belsey M J., Eur. J. Pharm. Biopharm. (2005) 59 (3),    389-396-   [Non-patent document 3] Monoclonal antibodies: versatile platforms    for cancer immunotherapy. Weiner L M, Surana R, Wang S., Nat. Rev.    Immunol. (2010) 10 (5), 317-327-   [Non-patent document 4] Differential responses of human tumor cell    lines to anti-p185HER2 monoclonal antibodies. Lewis G D, Figari I,    Fendly B, Wong W L, Carter P, Gorman C, Shepard H M, Cancer Immunol.    Immunotherapy (1993) 37, 255-263-   [Non-patent document 5] ING-1, a monoclonal antibody targeting    Ep-CAM in patients with advanced adenocarcinomas. de Bono J S,    Tolcher A W, Forero A, Vanhove G F, Takimoto C, Bauer R J, Hammond L    A, Patnaik A, White M L, Shen S, Khazaeli M B, Rowinsky E K,    LoBuglio A F, Clin. Cancer Res. (2004) 10 (22), 7555-7565-   [Non-patent document 6] Non-fucosylated therapeutic antibodies as    next-generation therapeutic antibodies. Satoh M, Iida S, Shitara K.,    Expert Opin. Biol. Ther. (2006) 6 (11), 1161-1173-   [Non-patent document 7] Optimizing engagement of the immune system    by anti-tumor antibodies: an engineer's perspective. Desjarlais J R,    Lazar G A, Zhukovsky E A, Chu S Y., Drug Discov. Today (2007) 12    (21-22), 898-910-   [Non-patent document 8] Antibody-drug conjugates: targeted drug    delivery for cancer. Alley S C, Okeley N M, Senter P D., Curr. Opin.    Chem. Biol. (2010) 14 (4), 529-537-   [Non-patent document 9] BiTE: Teaching antibodies to engage T-cells    for cancer therapy. Baeuerle P A, Kufer P, Bargou R., Curr. Opin.    Mol. Ther. (2009) 11 (1), 22-30-   [Non-patent document 10] T cell-engaging BiTE antibodies specific    for EGFR potently eliminate KRAS- and BRAF-mutated colorectal cancer    cells. Lutterbuese R, Raum T, Kischel R, Hoffmann P, Mangold S,    Rattel B, Friedrich M, Thomas 0, Lorenczewski G, Rau D, Schaller E,    Herrmann I, Wolf A, Urbig T, Baeuerle P A, Kufer P., Proc. Natl.    Acad. Sci. U.S.A. (2010) 107 (28), 12605-12610-   [Non-patent document 11] Phase I trial with the C D44v6-targeting    immunoconjugate bivatuzumab mertansine in head and neck squamous    cell carcinoma. Riechelmann H, Sauter A, Golze W, Hanft G, Schroen    C, Hoermann K, Erhardt T, Gronau S., Oral Oncol. (2008) 44 (9),    823-829-   [Non-patent document 12] Ipilimumab in the treatment of melanoma.    Trinh V A, Hwu W J., Expert Opin. Biol. Ther., (2012) April 14    (doi:10.1517/14712598.2012.675325)-   [Non-patent document 13] IPILIMUMAB—A NOVEL IMMUNOMODULATING THERAPY    CAUSING AUTOIMMUNE HYPOPHYSITIS: A CASE REPORT AND REVIEW. Juszczak    A, Gupta A, Karavitaki N, Middleton M R, Grossman A., Eur. J.    Endocrinol. (2012) April 10 (doi: 10.1530/EJE-12-0167)-   [Non-patent document 14] The Japanese experience with biologic    therapies for rheumatoid arthritis. Takeuchi T, Kameda H., Nat. Rev.    Rheumatol. (2010) 6 (11), 644-652-   [Non-patent document 15] Current evidence for the management of    rheumatoid arthritis with biological disease-modifying antirheumatic    drugs: a systematic literature review informing the EULAR    recommendations for the management of R A. Nam J L, Winthrop K L,    van Vollenhoven R F, Pavelka K, Valesini G, Hensor E M, Worthy G,    Landewe R, Smolen J S, Emery P, Buch M H., Ann Rheum. Dis. (2010) 69    (6), 976-986-   [Non-patent document 16] Antibody engineering for the development of    therapeutic antibodies. Kim S J, Park Y, Hong H J., Mol.    Cells. (2005) 20 (1), 17-29

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention was achieved in view of the above circumstances.An objective of the present invention is to provide pharmaceuticalcompositions that are useful for treating diseases originating fromtarget tissues, and active ingredients thereof. Another objective is toprovide methods of screening for the pharmaceutical compositions andactive ingredients, as well as their production methods.

Means for Solving the Problems

The present inventors conducted dedicated studies to achieve theabove-described objectives. As a result, they generated antigen-bindingmolecules comprising an antigen-binding domain whose antigen-bindingactivity varies depending on the concentration of the targettissue-specific compound. Furthermore, the present inventors discoveredthat the antigen-binding molecules or pharmaceutical compositionscomprising the antigen-binding molecules are useful for treatingdiseases that originate from a target tissue, and that they are alsouseful for treatment of diseases originating from target tissues thatincludes administering the antigen-binding molecules. They alsodiscovered that the antigen-binding molecules are useful in theproduction of pharmaceuticals for treating diseases that originate fromtarget tissues. Furthermore, the present inventors produced screeningmethods and production methods for the antigen-binding molecules, andthereby completed the present invention.

More specifically, the present invention provides the following:

[1] An antigen-binding molecule comprising an antigen-binding domainwhose antigen-binding activity varies depending on the concentration ofa target tissue-specific compound.[2] The antigen-binding molecule of [1], wherein the target tissue is acancer tissue.[3] The antigen-binding molecule of [2], wherein the compound specificto a cancer tissue is a metabolite specific to a cancer cell, ametabolite specific to an immune cell that has infiltrated into a cancertissue, or a metabolite specific to a stromal cell in a cancer tissue.[4] The antigen-binding molecule of [1], wherein the target tissue is aninflamed tissue.[5] The antigen-binding molecule of [4], wherein the compound specificto an inflamed tissue is a metabolite specific to an immune cell thathas infiltrated into an inflamed tissue or a metabolite specific to anormal cell that has been damaged in an inflamed tissue.[6] The antigen-binding molecule of [1], wherein the compound is atleast one compound selected from a nucleoside having a purine ringstructure, an amino acid and its metabolite, a lipid and its metabolite,a primary metabolite of glycometabolism, and nicotinamide and itsmetabolite.[7] The antigen-binding molecule of [6], wherein the compound is atleast one compound selected from adenosine, adenosine triphosphate,inosine, alanine, glutamic acid, aspartic acid, kynurenine,prostaglandin E2, succinic acid, citric acid, and 1-methylnicotinamide.[8] The antigen-binding molecule of any one of [1] to [7], wherein theantigen is a membrane-type molecule.[9] The antigen-binding molecule of any one of [1] to [8], which is anantigen-binding molecule that has a neutralizing activity.[10] The antigen-binding molecule of any one of [1] to [9], which is anantigen-binding molecule that has a cytotoxic activity.[11] The antigen-binding molecule of any one of [1] to [10], whichcomprises an Fc region.[12] The antigen-binding molecule of [11], wherein the Fc region is anFc region contained in the constant region of SEQ ID NOs: 5, 6, 7, or 8.[13] The antigen-binding molecule of [11], wherein the Fc regioncomprises an altered FcγR-binding Fc region that has a higher Fcγreceptor-binding activity than the Fcγ receptor-binding activity of anative human IgG Fc region.[14] The antigen-binding molecule of [13], wherein at least one or moreamino acids selected from the group consisting of amino acids atpositions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250,251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284,285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339,376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434,436, and 440 according to EU numbering, in the amino acid sequence ofthe altered FcγR-binding Fc region are different from the amino acids ofthe native human IgG Fc region.[15] The antigen-binding molecule of [14], which comprises at least oneor more amino acids selected from the group consisting of:Lys or Tyr for the amino acid at position 221;Phe, Trp, Glu, or Tyr for the amino acid at position 222;Phe, Trp, Glu, or Lys for the amino acid at position 223;Phe, Trp, Glu, or Tyr for the amino acid at position 224;Glu, Lys, or Trp for the amino acid at position 225;Glu, Gly, Lys, or Tyr for the amino acid at position 227;Glu, Gly, Lys, or Tyr for the amino acid at position 228;Ala, Glu, Gly, or Tyr for the amino acid at position 230;Glu, Gly, Lys, Pro, or Tyr for the amino acid at position 231;Glu, Gly, Lys, or Tyr for the amino acid at position 232;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 233;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 234;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 235;Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 236;Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 237;Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 238;Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,Val, Trp, or Tyr for the amino acid at position 239;Ala, Ile, Met, or Thr for the amino acid at position 240;Asp, Glu, Leu, Arg, Trp, or Tyr for the amino acid at position 241;Leu, Glu, Leu, Gln, Arg, Trp, or Tyr for the amino acid at position 243;His for the amino acid at position 244;Ala for the amino acid at position 245;Asp, Glu, His, or Tyr for the amino acid at position 246;Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acidat position 247;Glu, His, Gln, or Tyr for the amino acid at position 249;Glu or Gln for the amino acid at position 250;Phe for the amino acid at position 251;Phe, Met, or Tyr for the amino acid at position 254;Glu, Leu, or Tyr for the amino acid at position 255;Ala, Met, or Pro for the amino acid at position 256;Asp, Glu, His, Ser, or Tyr for the amino acid at position 258;Asp, Glu, His, or Tyr for the amino acid at position 260;Ala, Glu, Phe, Ile, or Thr for the amino acid at position 262;Ala, Ile, Met, or Thr for the amino acid at position 263;Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Trp, or Tyr for the amino acid at position 264;Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 265;Ala, Ile, Met, or Thr for the amino acid at position 266;Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val,Trp, or Tyr for the amino acid at position 267;Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trpfor the amino acid at position 268;Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, orTyr for the amino acid at position 269;Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyrfor the amino acid at position 270;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 271;Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, orTyr for the amino acid at position 272;Phe or Ile for the amino acid at position 273;Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 274;Leu or Trp for the amino acid at position 275;Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, orTyr for the amino acid at position 276;Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, or Trp for the amino acid at position 278;Ala for the amino acid at position 279;Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, or Tyr for the amino acid atposition 280;Asp, Lys, Pro, or Tyr for the amino acid at position 281;Glu, Gly, Lys, Pro, or Tyr for the amino acid at position 282;Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr for the amino acidat position 283;Asp, Glu, Leu, Asn, Thr, or Tyr for the amino acid at position 284;Asp, Glu, Lys, Gln, Trp, or Tyr for the amino acid at position 285;Glu, Gly, Pro, or Tyr for the amino acid at position 286;Asn, Asp, Glu, or Tyr for the amino acid at position 288;Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for the amino acid atposition 290;Asp, Glu, Gly, His, Ile, Gln, or Thr for the amino acid at position 291;Ala, Asp, Glu, Pro, Thr, or Tyr for the amino acid at position 292;Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyrfor the amino acid at position 293;Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, orTyr for the amino acid at position 294;Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 295;Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, orVal for the amino acid at position 296;Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 297;Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, orTyr for the amino acid at position 298;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Val, Trp, or Tyr for the amino acid at position 299;Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, or Trp for the amino acid at position 300;Asp, Glu, His, or Tyr for the amino acid at position 301;Ile for the amino acid at position 302;Asp, Gly, or Tyr for the amino acid at position 303;Asp, His, Leu, Asn, or Thr for the amino acid at position 304;Glu, Ile, Thr, or Tyr for the amino acid at position 305;Ala, Asp, Asn, Thr, Val, or Tyr for the amino acid at position 311;Phe for the amino acid at position 313;Leu for the amino acid at position 315;Glu, or Gln for the amino acid at position 317;His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr for the amino acid atposition 318;Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr forthe amino acid at position 320;Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, or Tyr for theamino acid at position 322;Ile for the amino acid at position 323;Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr forthe amino acid at position 324;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 325;Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, orTyr for the amino acid at position 326;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr,Val, Trp, or Tyr for the amino acid at position 327;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 328;Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 329;Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 330;Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr for theamino acid at position 331;Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 332;Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyrfor the amino acid at position 333;Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid at position 334;Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyrfor the amino acid at position 335;Glu, Lys, or Tyr for the amino acid at position 336;Glu, His, or Asn for the amino acid at position 337;Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr for the aminoacid at position 339;Ala or Val for the amino acid at position 376;Gly or Lys for the amino acid at position 377;Asp for the amino acid at position 378;Asn for the amino acid at position 379;Ala, Asn, or Ser for the amino acid at position 380;Ala, or Ile for the amino acid at position 382;Glu for the amino acid at position 385;Thr for the amino acid at position 392;Leu for the amino acid at position 396;Lys for the amino acid at position 421;Asn for the amino acid at position 427;Phe, or Leu for the amino acid at position 428;Met for the amino acid at position 429;Trp for the amino acid at position 434;Ile for the amino acid at position 436; and Gly, His, Ile, Leu, or Tyrfor the amino acid at position 440 according to EU numbering in theamino acid sequence of the altered FcγR-binding Fc region.[16] The antigen-binding molecule of [11], wherein the Fc region ismodified so that there is a higher proportion of Fc region bound by afucose-deficient sugar chain in a composition of sugar chain bound atposition 297, according to EU numbering, of the Fc region, or so thatthere is a higher proportion of Fc region with an added bisectingN-acetylglucosamine.[17] The antigen-binding molecule of any one of [11] and [13] to [16],wherein the FcRn-binding activity of the Fc region under an acidic pHrange condition is enhanced compared to the FcRn-binding activity of theFc region of SEQ ID NO: 5, 6, 7, or 8.[18] The antigen-binding molecule of [17], wherein the Fc region is anFc region with substitution of at least one or more amino acids selectedfrom the group consisting of amino acids at positions 238, 244, 245,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270,272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312,314, 316, 317, 318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376,377, 378, 380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424,427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447,according to EU numbering, in the amino acid sequence of the Fc regioncomprised in the constant region of SEQ ID NO: 5, 6, 7, or 8.[19] The antigen-binding molecule of [18], wherein the Fc regioncomprises at least one or more amino acids selected from the groupconsisting of:Leu for the amino acid at position 238;Leu for the amino acid at position 244;Arg for the amino acid at position 245;Pro for the amino acid at position 249;Gln or Glu for the amino acid at position 250;Arg, Asp, Glu, or Leu for the amino acid at position 251;Phe, Ser, Thr, or Tyr for the amino acid at position 252;Ser or Thr for the amino acid at position 254;Arg, Gly, Ile, or Leu for the amino acid at position 255;Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr for the amino acid at position256;Ala, Ile, Met, Asn, Ser, or Val for the amino acid at position 257;Asp for the amino acid at position 258;Ser for the amino acid at position 260;Leu for the amino acid at position 262;Lys for the amino acid at position 270;Leu, or Arg for the amino acid at position 272;Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for theamino acid at position 279;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr,Trp, or Tyr for the amino acid at position 283;Asn for the amino acid at position 285;Phe for the amino acid at position 286;Asn or Pro for the amino acid at position 288;Val for the amino acid at position 293,Ala, Glu, Gln, or Met for the amino acid at position 307;Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp for the amino acid atposition 311;Pro for the amino acid at position 309;Ala, Asp, or Pro for the amino acid at position 312;Ala or Leu for the amino acid at position 314;Lys for the amino acid at position 316;Pro for the amino acid at position 317;Asn or Thr for the amino acid at position 318;Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid at position332;Asn, Thr, or Trp for the amino acid at position 339;Pro for the amino acid at position 341;Glu, His, Lys, Gln, Arg, Thr, or Tyr for the amino acid at position 343;Arg for the amino acid at position 375;Gly, Ile, Met, Pro, Thr, or Val for the amino acid at position 376;Lys for the amino acid at position 377;Asp, Asn, or Val for the amino acid at position 378;Ala, Asn, Ser, or Thr for the amino acid at position 380;Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyrfor the amino acid at position 382;Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the amino acid at position385;Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr for the amino acid at position386;Ala, Arg, His, Pro, Ser, or Thr for the amino acid at position 387;Asn, Pro, or Ser for the amino acid at position 389;Asn for the amino acid at position 423;Asn for the amino acid at position 427;Leu, Met, Phe, Ser, or Thr for the amino acid at position 428;Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, orTyr for the amino acid at position 430;His or Asn for the amino acid at position 431;Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid at position 433;Ala, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid at position 434;Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the amino acid at position436;Lys, Leu, Thr, or Trp for the amino acid at position 438;Lys for the amino acid at position 440;Lys for the amino acid at position 442; andIle, Pro, or Thr for the amino acid at position 308;as indicated by EU numbering, in the amino acid sequence of the Fcregion comprised in the constant region of SEQ ID NO: 5, 6, 7, or 8.[20] The antigen-binding molecule of any one of [1] to [19], wherein theantigen-binding domain is a multispecific or a multiparatopicantigen-binding domain.[21] The antigen-binding molecule of [20], wherein an antigen bound byat least one of the antigen-binding domains is a membrane-type moleculeexpressed on a cancer cell membrane, and an antigen bound by at leastone of the antigen-binding domains is a membrane-type molecule expressedon an effector cell membrane.[22] The antigen-binding molecule of [21], wherein the effector cell isan NK cell, a macrophage, or a T cell.[23] The antigen-binding molecule of [21] or [22], wherein themembrane-type molecule expressed on an effector cell membrane is aTCR-constituting polypeptide, CD2, CD3, CD28, CD44, CD16, CD32, CD64, orNKG2D.[24] The antigen-binding molecule of [20], wherein an antigen bound byat least one of the antigen-binding domains is a membrane-type moleculeexpressed on a cancer cell membrane, and an antigen bound by at leastone of the antigen-binding domains is a cytotoxic substance.[25] The antigen-binding molecule of any one of [20] to [24], whereinthe antigen-binding molecule is an antibody fragment.[26] The antigen-binding molecule of any one of [1] to [24], wherein theantigen-binding molecule is an antibody.[27] The antigen-binding molecule of any one of [1] to [7], wherein theantigen is a soluble molecule.[28] The antigen-binding molecule of [27], which is an antigen-bindingmolecule having a neutralizing activity.[29] The antigen-binding molecule of [27] or [28], which comprises an Fcregion.[30] The antigen-binding molecule of [29], wherein the Fc region is anFc region comprised in the constant region of SEQ ID NO: 5, 6, 7, or 8.[31] The antigen-binding molecule of [29], wherein the FcRn-bindingactivity of the Fc region under an acidic pH range condition is enhancedcompared to the FcRn-binding activity of the Fc region comprised in theconstant region of SEQ ID NO: 5, 6, 7, or 8.[32] The antigen-binding molecule of [31], wherein the Fc region is anFc region with substitution of at least one or more amino acids selectedfrom the group consisting of amino acids at positions 238, 244, 245,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270,272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312,314, 316, 317, 318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376,377, 378, 380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424,427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447,according to EU numbering, in the amino acid sequence of the Fc regioncomprised in the constant region of SEQ ID NO: 5, 6, 7, or 8.[33] The antigen-binding molecule of [32], wherein the Fc regioncomprises at least one or more amino acids selected from the groupconsisting of:Leu for the amino acid at position 238;Leu for the amino acid at position 244;Arg for the amino acid at position 245;Pro for the amino acid at position 249;Gln or Glu for the amino acid at position 250;Arg, Asp, Glu, or Leu for the amino acid at position 251;Phe, Ser, Thr, or Tyr for the amino acid at position 252;Ser or Thr for the amino acid at position 254;Arg, Gly, Ile, or Leu for the amino acid at position 255;Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr for the amino acid at position256;Ala, Ile, Met, Asn, Ser, or Val for the amino acid at position 257;Asp for the amino acid at position 258;Ser for the amino acid at position 260;Leu for the amino acid at position 262;Lys for the amino acid at position 270;Leu, or Arg for the amino acid at position 272;Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for theamino acid at position 279;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr,Trp, or Tyr for the amino acid at position 283;Asn for the amino acid at position 285;Phe for the amino acid at position 286;Asn or Pro for the amino acid at position 288;Val for the amino acid at position 293,Ala, Glu, Gln, or Met for the amino acid at position 307;Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp for the amino acid atposition 311;Pro for the amino acid at position 309;Ala, Asp, or Pro for the amino acid at position 312;Ala or Leu for the amino acid at position 314;Lys for the amino acid at position 316;Pro for the amino acid at position 317;Asn or Thr for the amino acid at position 318;Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid at position332;Asn, Thr, or Trp for the amino acid at position 339;Pro for the amino acid at position 341;Glu, His, Lys, Gln, Arg, Thr, or Tyr for the amino acid at position 343;Arg for the amino acid at position 375;Gly, Ile, Met, Pro, Thr, or Val for the amino acid at position 376;Lys for the amino acid at position 377;Asp, Asn, or Val for the amino acid at position 378;Ala, Asn, Ser, or Thr for the amino acid at position 380;Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyrfor the amino acid at position 382;Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the amino acid at position385;Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr for the amino acid at position386;Ala, Arg, His, Pro, Ser, or Thr for the amino acid at position 387;Asn, Pro, or Ser for the amino acid at position 389;Asn for the amino acid at position 423;Asn for the amino acid at position 427;Leu, Met, Phe, Ser, or Thr for the amino acid at position 428;Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, orTyr for the amino acid at position 430;His or Asn for the amino acid at position 431;Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid at position 433;Ala, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid at position 434;Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the amino acid at position436;Lys, Leu, Thr, or Trp for the amino acid at position 438;Lys for the amino acid at position 440;Lys for the amino acid at position 442; andIle, Pro, or Thr for the amino acid at position 308;as indicated by EU numbering, in the amino acid sequence of the Fcregion comprised in the constant region of SEQ ID NO: 5, 6, 7, or 8.[34] The antigen-binding molecule of [29], wherein the FcRn-bindingactivity of the Fc region under a neutral pH range condition is enhancedcompared to the FcRn-binding activity of the Fc region comprised in theconstant region of SEQ ID NO: 5, 6, 7, or 8.[35] The antigen-binding molecule of [34], wherein the Fc region is anFc region with substitution of at least one or more amino acids selectedfrom the group consisting of amino acids at positions 237, 248, 250,252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307,308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384,385, 386, 387, 389, 424, 428, 433, 434, and 436 according to EUnumbering, in the amino acid sequence of the Fc region comprised in theconstant region of SEQ ID NO: 5, 6, 7, or 8.[36] The antigen-binding molecule of [35], wherein the Fc regioncomprises at least one or more amino acids selected from the groupconsisting of:Met for the amino acid at position 237;Ile for the amino acid at position 248;Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr for the amino acid atposition 250;Phe, Trp, or Tyr for the amino acid at position 252;Thr for the amino acid at position 254;Glu for the amino acid at position 255;Asp, Asn, Glu, or Gln for the amino acid at position 256;Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val for the amino acid atposition 257;His for the amino acid at position 258;Ala for the amino acid at position 265;Ala or Glu for the amino acid at position 286;His for the amino acid at position 289;Ala for the amino acid at position 297;Ala for the amino acid at position 303;Ala for the amino acid at position 305;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Val, Trp, or Tyr for the amino acid at position 307;Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr for the amino acid at position308;Ala, Asp, Glu, Pro, or Arg for the amino acid at position 309;Ala, His, or Ile for the amino acid at position 311;Ala or His for the amino acid at position 312;Lys or Arg for the amino acid at position 314;Ala, Asp, or His for the amino acid at position 315;Ala for the amino acid at position 317;Val for the amino acid at position 332;Leu for the amino acid at position 334;His for the amino acid at position 360;Ala for the amino acid at position 376;Ala for the amino acid at position 380;Ala for the amino acid at position 382;Ala for the amino acid at position 384;Asp or His for the amino acid at position 385;Pro for the amino acid at position 386;Glu for the amino acid at position 387;Ala or Ser for the amino acid at position 389;Ala for the amino acid at position 424;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 428;Lys for the amino acid at position 433;Ala, Phe, His, Ser, Trp, or Tyr for the amino acid at position 434; andHis, Ile, Leu, Phe, Thr, or Val for the amino acid at position 436as indicated by EU numbering, in the amino acid sequence of the Fcregion of SEQ ID NO: 5, 6, 7, or 8.[37] The antigen-binding molecule of any one of [29] and [31] to [36],wherein the Fc region has a higher binding activity to an inhibitory Fcγreceptor than to an activating Fcγ receptor.[38] The antigen-binding molecule of [37], wherein the inhibitory Fcγreceptor is human FcγRIIb.[39] The antigen-binding molecule of [37] or [38], wherein theactivating Fcγ receptor is human FcγRIa, human FcγRIIa (R), humanFcγRIIa (H), human FcγRIIIa (V), or human FcγRIIIa (F).[40] The antigen-binding molecule of any one of [37] to [39], whereinthe amino acid at position 238 or 328 (EU numbering) of the Fc regionincludes an amino acid that is different from the amino acid of thenative human IgG Fc region.[41] The antigen-binding molecule of [40], wherein the amino acid atposition 238 indicated by EU numbering in the Fc region is Asp or theamino acid at position 328 is Glu.[42] The antigen-binding molecule of [40] or [41], which comprises atleast one or more amino acids selected from the group consisting of:Asp for the amino acid at position 233;Trp or Tyr for the amino acid at position 234;Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr for the amino acid at position237;Asp for the amino acid at position 239;Ala, Gln, or Val for the amino acid at position 267;Asn, Asp, or Glu for the amino acid at position 268;Gly for the amino acid at position 271;Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser, or Thr for the amino acid atposition 326;Arg, Lys, or Met for the amino acid at position 330;Ile, Leu, or Met for the amino acid at position 323; andAsp for the amino acid at position 296 according to EU numbering, in theamino acid sequence of the Fc region.[43] The antigen-binding molecule of any one of [27] to [42], whereinthe antigen-binding molecule is an antibody.[44] A method for producing the antigen-binding molecule of any one of[1] to [43], which comprises selecting an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of atarget tissue-specific compound.[45] A method of screening for the antigen-binding molecule of any oneof [1] to [43], which comprises selecting an antigen-binding domainwhose antigen-binding activity varies depending on the concentration ofa target tissue-specific compound.[46] A pharmaceutical composition comprising the antigen-bindingmolecule of any one of [1] to [43].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that a small-molecule-switch antibody does not bind toantigens in a normal environment where the small molecules are notpresent, but binds to the antigens in the target tissue where the smallmolecules are present at a high concentration.

FIG. 2 shows that the small molecule functions as a switch by fittingbetween the anti-small-molecule antibody and the antigen. If the smallmolecule is absent, the antibody-antigen interaction is insufficient andthe antibody cannot bind to the antigen, but if the small molecule ispresent, the antibody can bind to the antigen by having the smallmolecule placed between the antibody and the antigen.

FIG. 3 is a figure showing the result of ELISA for the binding of theantibody to human IL-6. The vertical axis shows the absorbance valueswhich assess the binding activity to human IL-6 of each of theantibodies in the presence or absence of each of the small molecules.

FIG. 4 is a sensorgram showing the interaction between 4 μmol/L of humanIL-6 and A11 in the presence or absence of 100 μmol/L kynurenine.

FIG. 5 shows a graph that evaluates change in the response of binding toA11 immobilized onto Sensorchip CM5, when interaction is allowed to takeplace for 60 seconds with 1 μmol/L of IL-6 as the analyte. The verticalaxis shows change in the response (RU) before and after IL-6interaction, and the horizontal axis shows the concentration ofkynurenine (μmol/L) contained in the solution at that time.

FIG. 6 shows a graph that evaluates the response to H01 which has beenimmobilized onto Sensorchip CM5, when interaction is allowed to takeplace for 60 seconds with 1 μmol/L of IL-6 as the analyte. The verticalaxis shows change in the response (RU) before and after IL-6interaction, and the horizontal axis shows the concentration ofkynurenine contained in the solution (μmol/L) at that time.

FIG. 7 shows a graph that evaluates the response to IL-6 which has beenimmobilized onto Sensorchip CM5, when interaction is allowed to takeplace for 60 seconds with 0.1 μmol/L of A11 as the analyte. The verticalaxis shows change in the response (RU) before and after A11 interaction,and the horizontal axis shows the concentration of kynurenine containedin the solution (μmol/L).

FIG. 8 shows a graph obtained by allowing A11 to interact with IL-6immobilized on Sensorchip CM5 in the presence of 100 μmol/L kynurenine,and then observing the dissociation of A11 from IL6 in the presence of abuffer containing 100 μmol/L kynurenine or in the presence of a bufferthat does not contain kynurenine. In the figure, the vertical axis showsvalues normalized by defining the amount of A11 bound in the presence of100 μmol/L kynurenine as 100, and the horizontal axis shows the passageof time (in seconds) from the start of the interaction.

FIG. 9 shows a sensorgram obtained by allowing 800, 400, 200, 100, 50,or 25 nmol/L of kynurenine to interact with IL-6 immobilized on asensorchip. The vertical axis shows change in the amount of IL-6 boundby kynurenine (RU) (the response at the start of the interactionexperiment was defined as 0), and the horizontal axis shows the passageof time from the start of the interaction experiment.

FIG. 10 shows the structure of 2′-Adenosine-PEG-peptide which is anadenosine analog used for immunization of rabbits.

FIG. 11 shows the structure of 5′-Adenosine-PEG-peptide which is anadenosine analog used for immunization of rabbits.

FIG. 12 shows the structure of 2′-Adenosine-PEG-biotin produced bysubstituting biotin for the peptide portion of the adenosine analog usedfor immunization of rabbits.

FIG. 13 shows the structure of 5′-Adenosine-PEG-biotin produced bysubstituting biotin for the peptide portion of the adenosine analog usedfor immunization of rabbits.

FIG. 14 is a graph where the vertical axis shows the value(N_binding_(—)100) obtained by dividing the amount of binding in theinteraction of each antibody with 2′-Adenosine-PEG-biotin by the capturelevel (RU) of each antibody, and the horizontal axis shows the value(N_stability_(—)100) obtained by dividing the value obtained 60 secondsafter dissociation of 2′-Adenosine-PEG-biotin from each antibody afterinteraction with 2′-Adenosine-PEG-biotin by the capture level (RU) ofeach antibody.

FIG. 15A indicates sensorgrams of surface plasmon resonance-basedanalyses which show that clone SMB0002 binds to (interacts with)adenosine. The sensorgrams show the interactions between SMB0002 and theantigen at 7.81, 31.3, 125, and 500 nM in order from the bottom.

FIG. 15B indicates sensorgrams of surface plasmon resonance-basedanalyses which show that clone SMB0002 binds to (interacts with) ATP.The sensorgrams show the interactions between SMB0002 and the antigen at78.1, 313, 1250, and 5000 nM in order from the bottom.

FIG. 15C indicates sensorgrams of surface plasmon resonance-basedanalyses which show that clone SMB0089 binds to (interacts with)adenosine. The sensorgrams show the interactions between SMB0089 and theantigen at 7.81, 31.3, 125, and 500 nM in order from the bottom.

FIG. 15D indicates sensorgrams of surface plasmon resonance-basedanalyses which show that clone SMB0089 binds to (interacts with) ATP.The sensorgrams show the interactions between SMB00089 and the antigenat 78.1, 313, 1250, and 5000 nM in order from the bottom.

FIG. 15E indicates sensorgrams of surface plasmon resonance-basedanalyses which show that clone SMB0104 binds to (interacts with)adenosine. The sensorgrams show the interactions between SMB0104 and theantigen at 7.81, 31.3, and 500 nM in order from the bottom.

FIG. 15F indicates sensorgrams of surface plasmon resonance-basedanalyses which show that clone SMB0104 binds to (interacts with) ATP.The sensorgrams show the interactions between SMB0104 and the antigen at78.1, 313, 1250, and 5000 nM in order from the bottom.

FIG. 16 indicates sensorgrams of surface plasmon resonance-basedanalyses which show that clone SMB0171 binds to (interacts with) ATP.The sensorgrams show the interactions between SMB0171 and the antigen at5 and 50 μM in order from the bottom.

FIG. 17 indicates the results of competitive ELISA which shows thatclone SMB0002 binds to adenosine and ATP.

FIG. 18 shows a graph that assesses the inhibitive ability of ATPtowards binding of biotin-labeled antigens (a mixture of5′-Adenosine-PEG-biotin and ATP-PEG-biotin) by ATNLSA1-4_D12.

FIG. 19 shows the concept of a rationally designed antibody library thatcan obtain adenosine/ATP-switch antibodies against any antigen, whereinthe library is made from antibody variable region portions that contactwith the antigens such that adenosine or ATP is positioned between theantibody and antigen.

FIG. 20 shows the concept of an adenosine-immunized rabbit antibodylibrary which yields adenosine/ATP-switch antibodies against any antigenand in which adenosine or ATP is sandwiched between the antibody and theantigen.

FIG. 21 is a figure showing the result of ELISA for binding of theantibody to human IL-6. The vertical axis shows the binding activity ofeach antibody to human IL-6 depending on the presence or absence ofamino acids or amino acid metabolites (kynurenine, tryptophan,phenylalanine, anthranilic acid, 3-hydroxykynurenine, and kynurenicacid), presented as absorbance values at wavelength of 450 nm.

FIG. 22 is a figure showing the result of ELISA for binding of theantibody to human IL-6. The vertical axis shows the binding activity ofthe I6NMSC1-3_(—)#03 antibody to human IL-6 depending on the presence orabsence of each small molecule (ATP, adenosine, inosine, PGE2, succinicacid, lactic acid, kynurenine, and a small-molecule cocktail), presentedas specific activity values calculated from absorbance values atwavelength of 450 nm.

FIG. 23 is a figure showing the result of ELISA for binding of theantibody to human IL-6. The vertical axis shows the binding activity ofthe I6NMSC1-3_(—)#17 antibody to human IL-6 depending on the presence orabsence of each small molecule (ATP, adenosine, inosine, PGE2, succinicacid, lactic acid, kynurenine, and a small-molecule cocktail), presentedas specific activity values calculated from absorbance values atwavelength of 450 nm.

FIG. 24 is a figure showing the result of ELISA for binding of theantibody to HSA. The vertical axis shows the binding activity of theHSNMSC1-4_(—)#22 antibody to HSA depending on the presence or absence ofeach small molecule (ATP, adenosine, inosine, PGE2, succinic acid,lactic acid, kynurenine, and a small-molecule cocktail), presented asabsorbance values at wavelength of 450 nm.

FIG. 25 is a figure showing the result of ELISA performed on cloneI6DL2C5-4_(—)076, which was obtained from the rationally designedantibody library against human IL-6 in the presence or absence of ATPand/or adenosine at 1 mM. The vertical axis shows the absorbance valuewhich evaluates binding activity of the antibody to human IL-6. Resultsobtained when using M13KO7 Helper Phage are presented as the negativecontrol.

FIG. 26 is a figure showing the result of ELISA performed on cloneHSDL3C5-4_(—)015, which was obtained from the rationally designedantibody library against human serum albumin in the presence or absenceof ATP and/or adenosine at 1 mM. The vertical axis shows the absorbancevalue which assesses binding activity of the antibody to human serumalbumin. Results obtained when using M13KO7 Helper Phage are presentedas the negative control.

FIG. 27 is a figure showing the result of ELISA performed on clone6RAD2C1-4_(—)011 and 6RAD2C1-4_(—)076, which were obtained from therationally designed antibody library against human IL-6 receptor in thepresence or absence of ATP and/or adenosine (written as ADO) at 1 mM,and in the presence or absence of a small-molecule cocktail (SC). Thevertical axis shows absorbance values which assess the binding activityof the antibody to the human IL-6 receptor. Results obtained when usingM13KO7 Helper Phage are presented as the negative control.

FIG. 28 is a figure showing the result of ELISA for binding of clone6RNMSC1-2_F02 to human IL-6R. The vertical axis shows the absorbancevalues which assess the binding activity of the antibody to human IL-6Rin the presence or absence of each small molecule.

FIG. 29 is a figure showing the result of ELISA for binding of clone6RNMSC1-3_G02 to human IL-6R. The vertical axis shows the absorbancevalues which assess the binding activity of the antibody to human IL-6Rin the presence or absence of each small molecule.

FIG. 30 is a figure showing the result of ELISA for binding of anantibody to human IL-6R. The vertical axis shows the absorbance valueswhich assess the binding activity of the antibody to human IL-6R in thepresence or absence of each amino acid or amino acid metabolite.

FIG. 31 presents sensorgrams showing the interaction between6RNMSC1-2_F02 and 1 μmol/L IL-6R in the presence of 100 μmol/Lkynurenine, in the presence of 10 mmol/L ATP, and in the absence ofkynurenine and ATP. The solid line indicates the interaction in thepresence of kynurenine, the dotted line indicates the interaction in thepresence of ATP, and the dashed line indicates the interaction in theirabsence.

FIG. 32 is a graph obtained by allowing 6RNMSC1-2_F02 to interact withIL-6R immobilized on Sensorchip CM5 in the presence of 100 μmol/Lkynurenine, and then observing the dissociation of 6RNMSC1-2_F02 fromIL-6R in the presence of a buffer containing 100 μmol/L kynurenine or inthe presence of a buffer that does not contain kynurenine. In thefigure, the vertical axis shows values normalized by defining the amountof 6RNMSC1-2_F02 bound in the presence of 100 μmol/L kynurenine as 100,and the horizontal axis shows the passage of time (in seconds) from thestart of the interaction. The solid line shows the dissociation of6RNMSC1-2_F02 from IL-6R in the presence of kynurenine, and the dottedline shows the dissociation of 6RNMSC1-2_F02 from IL-6R in the absenceof kynurenine.

FIG. 33 is a graph produced by allowing 5 μg/L of 6RNMSC1-2_F02 tointeract as an analyte for 180 seconds, and assessing the response toIL-6R immobilized onto Sensorchip CM5. The vertical axis shows change inthe response (RU) before and after 6RNMSC1-2_F02 interaction, and thehorizontal axis shows the concentration (μmol/L) of kynurenine containedin the solution.

FIG. 34 is a figure showing assessment of the binding of antibodies tomembrane-type human IL-6R by FCM. The top panel shows results obtainedin the presence of Kynurenine, and the bottom panel shows resultsobtained in the absence of Kynurenine. The horizontal axis shows thefluorescence intensity and the vertical axis shows the cell count.

FIG. 35A shows the ADCC activity of antibodies that bind to antigens inthe presence of small molecules toward cells expressing the antigens. Itshows the ADCC activity of clone 6RNMSC1-2_F02, which binds to hIL-6R inthe presence of kynurenine, toward BaF cells expressing hIL-6R in thepresence (triangles) or absence (circles) of kynurenine. The opentriangles and circles show the measured values, and the filled trianglesand circles show the mean values.

FIG. 35B shows the ADCC activity of antibodies that bind to antigens inthe presence of small molecules toward cells that express the antigen.It shows the ADCC activity of MRA, which binds to hIL-6R regardless ofthe presence of kynurenine, toward BaF cells expressing hIL-6R in thepresence (triangles) or absence (circles) of kynurenine. The opentriangles and circles show the measured values, and the filled trianglesand circles show the mean values.

FIG. 36 shows the ADCC activity of antibodies that bind to antigens inthe presence of small molecules toward cells expressing the antigen. Itshows the ADCC activity of clone 6RNMSC1-2_F02 toward BaF cellsexpressing hIL-6R in the presence (triangles) or absence (circles) ofclone 6RNMSC1-2_F02 which binds to hIL-6R in the presence of kynurenine.The horizontal axis shows the kynurenine concentration and the verticalaxis shows the ADCC activity (%). The mean values and standarddeviations of ADCC activity are shown.

FIG. 37 is a figure showing the result of ELISA for the binding of clone6RNMSC1-2_F02 in mouse serum to human IL-6R. The vertical axis shows theabsorbance values which evaluate the binding activities of the antibodyto human IL-6R in the presence or absence of kynurenine.

FIG. 38 is a figure showing the result of ELISA performed with cloneI6RLSA1-6_(—)011, which was obtained from the rationally designedantibody library, against human IL-6 in the presence or absence of ATPand adenosine at 10 mM. The vertical axis shows the absorbance valuewhich evaluates binding activity of the antibody to human IL-6. Resultsobtained when using a clone obtained from the rationally designedantibody library and showing binding activity toward human IL-6regardless of the presence of small molecules are presented as thepositive control. Results obtained when using the M13KO7 Helper Phageare presented as the negative control.

FIG. 39 is a figure showing the result of ELISA performed with clone6RRLSA1-6_(—)037 and 6RRLSA1-6_(—)045, which were obtained from therationally designed antibody library, against the human IL-6 receptor inthe presence or absence of ATP and adenosine at 10 mM. The vertical axisshows the absorbance value which evaluates the binding activity of theantibodies to the human IL-6 receptor. Results obtained when using theM13KO7 Helper Phage are presented as the negative control.

FIG. 40 is a figure showing the result of ELISA performed on 96 clonesobtained by panning the rationally designed antibody library four timesagainst human IgA-Fc using a multivalent antibody phage display. Theabsorbance values which evaluate the binding activity of the antibodiesto human IgA-Fc in the absence of ATP and adenosine are shown on thevertical axis, and absorbance values which evaluate the binding activityof the antibodies to human IgA-Fc in the presence of ATP and adenosineare shown on the horizontal axis.

FIG. 41 is a figure showing the result of ELISA performed on 96 clonesobtained by panning the rationally designed antibody library four timesagainst human IgA-Fc using a monovalent antibody phage display. Theabsorbance values which evaluate the binding activity of the antibodiesto human IgA-Fc in the absence of ATP and adenosine are shown on thevertical axis, and the absorbance values which evaluate the bindingactivity of the antibodies to human IgA-Fc in the presence of ATP andadenosine are shown on the horizontal axis.

FIG. 42 is a figure showing the result of ELISA performed on cloneIADL3C5-4_(—)048 obtained from the rationally designed antibody libraryagainst human IgA-Fc in the presence or absence of ATP and adenosine at1 mM. The vertical axis shows the absorbance value which evaluatesbinding activity of the antibody to human IgA-Fc. Results obtained whenusing a clone obtained from the rationally designed antibody library andshowing binding activity toward human IgA-Fc regardless of the presenceof small molecules are presented as the positive control. Resultsobtained when using the M13KO7 Helper Phage are presented as thenegative control.

FIG. 43 is a graph showing the binding level (binding response (RU))when each clone at 1 μM was made to interact for 120 seconds with IL-6Rimmobilized on Sensorchip CM5 in the presence or absence of each of thesmall molecules at 1 mM.

FIG. 44A shows the ADCC activity of antibodies that bind to antigens inthe presence of small molecules toward cells expressing the antigen. Itis a figure showing the ADCC activity of clone 6RAD2C1-4_(—)030, whichbinds to hIL-6R in the presence of ATP, toward CHO cells expressinghIL-6R in the presence (triangles) or absence (circles) of ATP. The opentriangles and circles show the measured values, and the filled trianglesand circles show the mean values.

FIG. 44B shows the ADCC activity of antibodies that bind to antigens inthe presence of small molecules toward cells expressing the antigen. Itis a figure showing the ADCC activity of clone 6RAD2C1-4_(—)011, whichbinds to hIL-6R in the presence of ATP, toward CHO cells expressinghIL-6R in the presence (triangles) or absence (circles) of ATP. The opentriangles and circles show the measured values, and the filled trianglesand circles show the mean values.

FIG. 44C shows the ADCC activity of antibodies that bind to antigens inthe presence of small molecules toward cells expressing the antigen. Itis a figure showing the ADCC activity of MRA, which binds to hIL-6Rregardless of the presence or absence of ATP, toward CHO cellsexpressing in the presence (triangles) or absence (circles) of ATP. Theopen triangles and circles show the measured values, and the filledtriangles and circles show the mean values.

FIG. 45 is a figure showing the result of ELISA performed on cloneHSADSA1-6_(—)020 obtained from the rationally designed antibody libraryagainst HSA in the presence or absence of ATP and adenosine at 10 mM.The vertical axis shows the absorbance value which evaluates bindingactivity of the antibody to HSA. Results obtained when using a cloneobtained from the rationally designed antibody library and showingbinding activity toward HSA regardless of the presence of smallmolecules are presented as the positive control. Results obtained whenusing the M13KO7 Helper Phage are presented as the negative control.

MODE FOR CARRYING OUT THE INVENTION

The definitions and detailed description below are provided tofacilitate understanding of the present invention illustrated herein.

Amino Acids

Herein, amino acids are described by one- or three-letter codes or both,for example, Ala/A, Leu/L, Arg/R, Lys/K, Asn/N, Met/M, Asp/D, Phe/F,Cys/C, Pro/P, Gln/Q, Ser/S, Glu/E, Thr/T, Gly/G, Trp/W, His/H, Tyr/Y,Ile/I, or Val/V.

Alteration of Amino Acids

For amino acid alteration in the amino acid sequence of anantigen-binding molecule, known methods such as site-directedmutagenesis methods (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985)82, 488-492)) and overlap extension PCR may be appropriately employed.Furthermore, several known methods may also be employed as amino acidalteration methods for substitution to non-natural amino acids (AnnuRev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad.Sci. U.S.A. (2003) 100 (11), 6353-6357). For example, it is suitable touse a cell-free translation system (Clover Direct (Protein Express))containing a tRNA which has a non-natural amino acid bound to acomplementary amber suppressor tRNA of one of the stop codons, the UAGcodon (amber codon).

In the present specification, the meaning of the term “and/or” whendescribing the site of amino acid alteration includes every combinationwhere “and” and “or” are suitably combined. Specifically, for example,“the amino acids at positions 33, 55, and/or 96 are substituted”includes the following variation of amino acid alterations: aminoacid(s) at (a) position 33, (b) position 55, (c) position 96, (d)positions 33 and 55, (e) positions 33 and 96, (f) positions 55 and 96,and (g) positions 33, 55, and 96.

Furthermore, herein, as an expression showing alteration of amino acids,an expression that shows before and after a number indicating a specificposition, one-letter or three-letter codes for amino acids before andafter alteration, respectively, may be used appropriately. For example,the alteration N100bL or Asn100bLeu used when substituting an amino acidcontained in an antibody variable region indicates substitution of Asnat position 100b (according to Kabat numbering) with Leu. That is, thenumber shows the amino acid position according to Kabat numbering, theone-letter or three-letter amino-acid code written before the numbershows the amino acid before substitution, and the one-letter orthree-letter amino-acid code written after the number shows the aminoacid after substitution. Similarly the alteration P238D or Pro238Aspused when substituting an amino acid of the Fc region contained in anantibody constant region indicates substitution of Pro at position 238(according to EU numbering) with Asp. That is, the number shows theamino acid position according to EU numbering, the one-letter orthree-letter amino-acid code written before the number shows the aminoacid before substitution, and the one-letter or three-letter amino-acidcode written after the number shows the amino acid after substitution.

Antigens

Herein, “antigens” are not particularly limited in their structure, aslong as they comprise epitopes to which antigen-binding domains bind. Inother words, antigens can be inorganic or organic substances. Otherantigens include, for example, the molecules below: 17-IA, 4-1BB, 4Dc,6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE,ACE-2, activin, activin A, activin AB, activin B, activin C, activinRIA, activin RIAALK-2, activin RIB ALK-4, activin RIIA, activin RIIB,ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMS, ADAM9, ADAMTS,ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE,APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrialnatriuretic peptide, av/b3 integrin, Ax1, b2M, B7-1, B7-2, B7-H,B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R,Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik,BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR,BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP,b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA,BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer associatedantigen, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D,cathepsin E, cathepsin H, cathepsin L, cathepsin 0, cathepsin S,cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC,CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4,PD1, PDL1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2,CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12,CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5,CXCR6, cytokeratin tumor associated antigen, DAN, DCC, DcR3, DC-SIGN,complement regulatory factor (Decay accelerating factor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26,Dtk, EGAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN,ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM,ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII,factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1,FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin,FL, FLIP, Flt-3, Flt-4, follicle stimulating hormone, fractalkine, FZD1,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6,GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14,CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8(myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1,GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, glucagon, Glut4, glycoproteinIIb/IIIa (GPIIb/IIIa), GM-CSF, gp130, gp72, GRO, growth hormonereleasing hormone, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBenvelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL,hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2,Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus(HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM 1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon(INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain,insulin B chain, insulin-like growth factor1, integrin alpha2, integrinalpha3, integrin alpha4, integrin alpha4/beta1, integrin alpha4/beta7,integrin alpha5 (alpha V), integrin alpha5/beta1, integrin alpha5/beta3,integrin alpha6, integrin beta1, integrin beta2, interferon gamma,IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1,kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocytegrowth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1,latent TGF-1 bpl, LBP, LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Yassociated antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX,LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface,luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG,MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES,MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK,MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15,MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin(Muc1), MUC18, Mullerian-inhibiting substance, Mug, MuSK, NAIP, NAP,NCAD, N-C adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4,or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO,NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150,p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD,P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2,PGD2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14,proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specificmembrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL,RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratorysyncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factor, RLIP76, RPA2,RSK, 5100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh, SIGIRR,SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II,TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3,T-cell receptor (for example, T-cell receptor alpha/beta), TdT, TECK,TEM1, TEM5, TEM7, TEM8, TERT, testis PLAP-like alkaline phosphatase,TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-betaRI(ALK-5), TGF-betaRII, TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2,TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymus Ck-1,thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2,Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI,TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5,KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRill, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand,TL2), TNFSF11 (TRANCE/RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-α Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferrin receptor, TRF, Trk, TROP-2, TLR1 (Toll-like receptor 1),TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumorassociated antigen CA125, tumor associated antigen expressing Lewis-Yassociated carbohydrates, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase,VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (fit-1), VEGF,VEGFR, VEGFR-3 (flt-4), VEGI, VIM, virus antigen, VLA, VLA-1, VLA-4, VNRintegrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3,WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A,WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1,XEDAR, XIAP, XPD, HMGB1, IgA, AP, CD81, CD97, CD98, DDR1, DKK1, EREG,Hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9, prekallikrein,RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, highmolecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3,Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s,C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9,factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin,prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII,factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X,factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII,factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA,plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2,Syndecan-3, Syndecan-4, LPA, and S1P; and receptors for hormone andgrowth factors. Preferred antigens are antigens that are expressed incancer cells, immune cells, stromal cells, or such present in cancertissues or inflammatory tissues.

While receptors are recited as examples of the above-mentioned antigens,when these receptors exist in soluble forms in biological fluids, theymay be used as antigens that bind to the antigen-binding molecule of thepresent invention, which contains an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of thetarget tissue-specific compound. An example of a non-limiting embodimentof such a soluble receptor is the soluble IL-6R, which is a proteinconsisting of the amino acids at positions 1 to 357 in the IL-6Rpolypeptide sequence of SEQ ID NO: 1 as described in Mullberg et al. (J.Immunol. (1994) 152 (10), 4958-4968).

Membrane-type molecules expressed on cell membranes and solublemolecules secreted from cells to the outside of the cells are includedin the examples of the above-mentioned antigens. When theantigen-binding molecule of the present invention, which contains anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of the target tissue-specific compound, binds to asoluble molecule secreted from cells, it is preferable that theantigen-binding molecule has neutralizing activity as described later.

The fluids in which the soluble molecules exist are not limited, and thesoluble molecules may exist in biological fluids, or more specificallyin all fluids filling the space between tissues and cells or vessels inorganisms. In a non-limiting embodiment, the soluble molecules to whichantigen-binding molecules of the present invention bind may be presentin the extracellular fluid. In vertebrates, extracellular fluid is ageneral term for plasma, interstitial fluid, lymph, compact connectivetissue, cerebrospinal fluid, spinal fluid, puncture fluid, synovialfluid, or such components in the bone and cartilage, alveolar fluid(bronchoalveolar lavage fluid), peritoneal fluid, pleural fluid,pericardial effusion, cyst fluid, aqueous humor (hydatoid), or suchtranscellular fluids (various fluids in the glandular cavities andfluids in the digestive tract cavity and other body cavity fluidsproduced as a result of active transport/secretory activities of cells).

When an antigen-binding molecule of the present invention comprising anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a target tissue-specific compound binds to amembrane-type molecule expressed on a cell membrane, suitable examplesof the antigen-binding molecule include antigen-binding molecules whichhave cytotoxic activity, bind to a cytotoxic substance, or have theability to bind to a cytotoxic substance, as described later.Furthermore, antigen-binding molecules having a neutralizing activityinstead of the properties of having a cytotoxic activity, binding to acytotoxic substance, or having the ability to bind to a cytotoxicsubstance; or in addition to these properties are also suitable examplesof a non-limiting embodiment.

Epitopes

“Epitope” means an antigenic determinant in an antigen, and refers to anantigen site to which the antigen-binding domain of an antigen-bindingmolecule disclosed herein binds. Thus, for example, the epitope can bedefined according to its structure. Alternatively, the epitope may bedefined according to the antigen-binding activity of an antigen-bindingmolecule that recognizes the epitope. When the antigen is a peptide orpolypeptide, the epitope can be specified by the amino acid residuesforming the epitope. Alternatively, when the epitope is a sugar chain,the epitope can be specified by its specific sugar chain structure.

A linear epitope is an epitope that contains an epitope whose primaryamino acid sequence has been recognized. Such a linear epitope typicallycontains at least three and most commonly at least five, for example,about 8 to about 10 or 6 to 20 amino acids in a specific sequence.

In contrast to the linear epitope, a “conformational epitope” is anepitope in which the primary amino acid sequence containing the epitopeis not the only determinant of the recognized epitope (for example, theprimary amino acid sequence of a conformational epitope is notnecessarily recognized by an epitope-defining antibody). Conformationalepitopes may contain a greater number of amino acids compared to linearepitopes. A conformational epitope-recognizing antibody recognizes thethree-dimensional structure of a peptide or protein. For example, when aprotein molecule folds and forms a three-dimensional structure, aminoacids and/or polypeptide main chains that form a conformational epitopebecome aligned, and the epitope is made recognizable by the antibody.Methods for determining epitope conformations include, for example, Xray crystallography, two-dimensional nuclear magnetic resonance,site-specific spin labeling, and electron paramagnetic resonance, butare not limited thereto. See, for example, Epitope Mapping Protocols inMethods in Molecular Biology (1996), Vol. 66, Morris (ed.).

The structure of the antigen-binding domain which binds to an epitope iscalled a paratope. An epitope and a paratope bind with stability throughthe action of hydrogen bonds, electrostatic force, van der Waals force,hydrophobic bonds, and such between the epitope and the paratope. Thisstrength of binding between the epitope and paratope is called affinity.The total sum of binding strength when a plurality of antigens and aplurality of antigen-binding molecules bind is referred to as avidity.When an antibody comprising a plurality of antigen-binding domains(i.e., multivalent antibody) or such binds to a plurality of epitopes,the affinity acts synergistically, and therefore avidity becomes higherthan affinity.

Binding Activity

Examples of a method for assessing the epitope binding by a testantigen-binding molecule containing an IL-6R antigen-binding domain aredescribed below. According to the examples below, methods for assessingthe epitope binding by a test antigen-binding molecule containing anantigen-binding domain for an antigen other than IL-6R, can also beappropriately conducted.

For example, whether a test antigen-binding molecule containing an IL-6Rantigen-binding domain recognizes a linear epitope in the IL-6R moleculecan be confirmed for example as mentioned below. A linear peptidecomprising an amino acid sequence forming the extracellular domain ofIL-6R is synthesized for the above purpose. The peptide can besynthesized chemically, or obtained by genetic engineering techniquesusing a region encoding the amino acid sequence corresponding to theextracellular domain in an IL-6R cDNA. Then, a test antigen-bindingmolecule containing an IL-6R antigen-binding domain is assessed for itsbinding activity towards a linear peptide comprising the amino acidsequence forming the extracellular domain. For example, an immobilizedlinear peptide can be used as an antigen by ELISA to evaluate thebinding activity of the antigen-binding molecule towards the peptide.Alternatively, the binding activity towards a linear peptide can beassessed based on the level that the linear peptide inhibits the bindingof the antigen-binding molecule to IL-6R-expressing cells. These testscan demonstrate the binding activity of the antigen-binding moleculetowards the linear peptide.

Whether a test antigen-binding molecule containing an IL-6Rantigen-binding domain recognizes a conformational epitope can beassessed as follows. IL-6R-expressing cells are prepared for the abovepurpose. A test antigen-binding molecule containing an IL-6Rantigen-binding domain can be determined to recognize a conformationalepitope when it strongly binds to IL-6R-expressing cells upon contact,but does not substantially bind to an immobilized linear peptidecomprising an amino acid sequence forming the extracellular domain ofIL-6R. Herein, “not substantially bind” means that the binding activityis 80% or less, generally 50% or less, preferably 30% or less, andparticularly preferably 15% or less compared to the binding activitytowards cells expressing human IL-6R.

Methods for assaying the binding activity of a test antigen-bindingmolecule containing an IL-6R antigen-binding domain towardsIL-6R-expressing cells include, for example, the methods described inAntibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold SpringHarbor Laboratory (1988) 359-420). Specifically, the assessment can beperformed based on the principle of ELISA or fluorescence activated cellsorting (FACS) using IL-6R-expressing cells as antigen.

In the ELISA format, the binding activity of a test antigen-bindingmolecule containing an IL-6R antigen-binding domain towardsIL-6R-expressing cells can be assessed quantitatively by comparing thelevels of signal generated by enzymatic reaction. Specifically, a testpolypeptide complex is added to an ELISA plate onto whichIL-6R-expressing cells are immobilized. Then, the test antigen-bindingmolecule bound to the cells is detected using an enzyme-labeled antibodythat recognizes the test antigen-binding molecule. Alternatively, whenFACS is used, a dilution series of a test antigen-binding molecule isprepared, and the antibody binding titer for IL-6R-expressing cells canbe determined to compare the binding activity of the testantigen-binding molecule towards IL-6R-expressing cells.

The binding of a test antigen-binding molecule towards an antigenexpressed on the surface of cells suspended in buffer or the like can bedetected using a flow cytometer. Known flow cytometers include, forexample, the following devices:

FACSCanto™ II FACSAria™ FACSArray™ FACSVantage™ SE

FACSCalibur™ (all are trade names of BD Biosciences)

EPICS ALTRA HyPerSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC

Cell Lab Quanta/Cell Lab Quanta SC (all are trade names of BeckmanCoulter).

Preferable methods for assaying the binding activity of a testantigen-binding molecule containing an IL-6R antigen-binding domaintowards an antigen include, for example, the following method. First,IL-6R-expressing cells are reacted with a test antigen-binding molecule,and then this is stained with an FITC-labeled secondary antibody thatrecognizes the antigen-binding molecule. The test antigen-bindingmolecule is appropriately diluted with a suitable buffer to prepare themolecule at a desired concentration. For example, the molecule can beused at a concentration within the range of 10 μg/ml to 10 ng/ml. Then,the fluorescence intensity and cell count are determined usingFACSCalibur (BD). The fluorescence intensity obtained by analysis usingthe CELL QUEST Software (BD), i.e., the Geometric Mean value, reflectsthe quantity of antibody bound to cells. That is, the binding activityof a test antigen-binding molecule, which is represented by the quantityof the test antigen-binding molecule bound, can be determined bymeasuring the Geometric Mean value.

Whether a test antigen-binding molecule containing an IL-6Rantigen-binding domain shares a common epitope with anotherantigen-binding molecule can be assessed based on the competitionbetween the two molecules for the same epitope. The competition betweenantigen-binding molecules can be detected by cross-blocking assay or thelike. For example, the competitive ELISA assay is a preferredcross-blocking assay.

Specifically, in cross-blocking assay, the IL-6R protein immobilized tothe wells of a microtiter plate is pre-incubated in the presence orabsence of a candidate competitor antigen-binding molecule, and then atest antigen-binding molecule is added thereto. The quantity of testantigen-binding molecule bound to the IL-6R protein in the wells isindirectly correlated with the binding ability of a candidate competitorantigen-binding molecule that competes for the binding to the sameepitope. That is, the greater the affinity of the competitorantigen-binding molecule for the same epitope, the lower the bindingactivity of the test antigen-binding molecule towards the IL-6Rprotein-coated wells.

The quantity of the test antigen-binding molecule bound to the wells viathe IL-6R protein can be readily determined by labeling theantigen-binding molecule in advance. For example, a biotin-labeledantigen-binding molecule is measured using an avidin/peroxidaseconjugate and appropriate substrate. In particular, cross-blocking assaythat uses enzyme labels such as peroxidase is called “competitive ELISAassay”. The antigen-binding molecule can also be labeled with otherlabeling substances that enable detection or measurement. Specifically,radiolabels, fluorescent labels, and such are known.

When the candidate competitor antigen-binding molecule can block thebinding by a test antigen-binding molecule containing an IL-6Rantigen-binding domain by at least 20%, preferably at least 20 to 50%,and more preferably at least 50% compared to the binding activity in acontrol experiment conducted in the absence of the competitorantigen-binding molecule, the test antigen-binding molecule isdetermined to substantially bind to the same epitope bound by thecompetitor antigen-binding molecule, or compete for the binding to thesame epitope.

When the structure of an epitope bound by a test antigen-bindingmolecule containing an IL-6R antigen-binding domain has already beenidentified, whether the test and control antigen-binding molecules sharea common epitope can be assessed by comparing the binding activities ofthe two antigen-binding molecules towards a peptide prepared byintroducing amino acid mutations into the peptide forming the epitope.

To measure the above binding activities, for example, the bindingactivities of test and control antigen-binding molecules towards alinear peptide into which a mutation is introduced are compared in theabove ELISA format. Besides the ELISA methods, the binding activitytowards the mutant peptide bound to a column can be determined byflowing test and control antigen-binding molecules in the column, andthen quantifying the antigen-binding molecule eluted in the elutionsolution. Methods for adsorbing a mutant peptide to a column, forexample, in the form of a GST fusion peptide, are known.

Alternatively, when the identified epitope is a conformational epitope,whether test and control antigen-binding molecules share a commonepitope can be assessed by the following method. First, IL-6R-expressingcells and cells expressing IL-6R with a mutation introduced into theepitope are prepared. The test and control antigen-binding molecules areadded to a cell suspension prepared by suspending these cells in anappropriate buffer such as PBS. Then, the cell suspensions areappropriately washed with a buffer, and an FITC-labeled antibody thatrecognizes the test and control antigen-binding molecules is addedthereto. The fluorescence intensity and number of cells stained with thelabeled antibody are determined using FACSCalibur (BD). The test andcontrol antigen-binding molecules are appropriately diluted using asuitable buffer, and used at desired concentrations. For example, theymay be used at a concentration within the range of 10 μg/ml to 10 ng/ml.The fluorescence intensity determined by analysis using the CELL QUESTSoftware (BD), i.e., the Geometric Mean value, reflects the quantity oflabeled antibody bound to cells. That is, the binding activities of thetest and control antigen-binding molecules, which are represented by thequantity of labeled antibody bound, can be determined by measuring theGeometric Mean value.

In the above method, whether an antigen-binding molecule does “notsubstantially bind to cells expressing mutant IL-6R” can be assessed,for example, by the following method. First, the test and controlantigen-binding molecules bound to cells expressing mutant IL-6R arestained with a labeled antibody. Then, the fluorescence intensity of thecells is determined. When FACSCalibur is used for fluorescence detectionby flow cytometry, the determined fluorescence intensity can be analyzedusing the CELL QUEST Software. From the Geometric Mean values in thepresence and absence of the polypeptide complex, the comparison value(ΔGeo-Mean) can be calculated according to Formula 1 below to determinethe ratio of increase in fluorescence intensity as a result of thebinding by the antigen-binding molecule.

ΔGeo-Mean=Geo-Mean (in the presence of the polypeptide complex)/Geo-Mean(in the absence of the polypeptide complex)  Formula 1

The Geometric Mean comparison value (ΔGeo-Mean value for the mutantIL-6R molecule) determined by the above analysis, which reflects thequantity of a test antigen-binding molecule bound to cells expressingmutant IL-6R, is compared to the ΔGeo-Mean comparison value thatreflects the quantity of the test antigen-binding molecule bound toIL-6R-expressing cells. In this case, the concentrations of the testantigen-binding molecule used to determine the ΔGeo-Mean comparisonvalues for IL-6R-expressing cells and cells expressing mutant IL-6R areparticularly preferably adjusted to be equal or substantially equal. Anantigen-binding molecule that has been confirmed to recognize an epitopein IL-6R is used as a control antigen-binding molecule.

If the ΔGeo-Mean comparison value of a test antigen-binding molecule forcells expressing mutant IL-6R is smaller than the ΔGeo-Mean comparisonvalue of the test antigen-binding molecule for IL-6R-expressing cells byat least 80%, preferably 50%, more preferably 30%, and particularlypreferably 15%, then the test antigen-binding molecule “does notsubstantially bind to cells expressing mutant IL-6R”. The formula fordetermining the Geo-Mean (Geometric Mean) value is described in the CELLQUEST Software User's Guide (BD biosciences). When the comparison showsthat the comparison values are substantially equivalent, the epitope forthe test and control antigen-binding molecules can be determined to bethe same.

Target Tissue

The term “target tissue” as used herein refers to a tissue containingcells carrying antigens to which the antigen-binding molecules of thepresent invention bind in a manner dependent on compounds. It is atissue that yields positive pharmacological effects for the organismcarrying the tissue, when the antigen-binding molecules bind to amembrane-type molecule expressed on the cells or bind to a solublemolecule present in the tissue. In this case, the phrase “positivepharmacological effects” refers to effects that relieve, alleviate,ameliorate, or cure symptoms brought about by pathological sitescontaining the target tissue for the organism carrying the tissue. Whenthe symptoms are brought about by malignant tumors such as cancer, anon-limiting embodiment of a mechanism that yields such apharmacological effect is, for example, cytotoxic activity and growthinhibition against cancer cells, and immunostimulation in cancertissues. In the case of inflammatory diseases, examples of such anon-limiting embodiment of the mechanism include immunosuppression andactivity to block actions of inflammatory cytokines in inflammatorytissues.

Cancer Tissue-Specific Compounds

The term “compound specific to a cancer tissue (cancer tissue-specificcompound)” as used herein refers to a compound differentially present incancer tissues as compared to non-cancerous tissues. Herein, the term“cancer” is generally used to describe malignant neoplasms, which may bemetastatic or non-metastatic. Non-limiting examples of carcinomasdeveloped from epithelial tissues such as skin or digestive tractinclude brain tumor, skin cancer, head and neck cancer, esophagealcancer, lung cancer, stomach cancer, duodenal cancer, breast cancer,prostate cancer, cervical cancer, endometrial cancer, pancreatic cancer,liver cancer, colorectal cancer, colon cancer, bladder cancer, andovarian cancer. Non-limiting examples of sarcomas developed fromnon-epithelial (interstitial) tissues such as muscles includeosteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma,liposarcoma, and angiosarcoma. Non-limiting examples of hematologicalcancer derived from hematopoietic organs include malignant lymphomasincluding Hodgkin's lymphoma and non Hodgkin's lymphoma; leukemiaincluding acute myelocytic leukemia or chronic myelocytic leukemia, andacute lymphatic leukemia or chronic lymphatic leukemia; and multiplemyeloma. The term “neoplasm” widely used herein refers to any newlyformed diseased tissue tumor. In the present invention, neoplasms causeformation of tumors, which are partly characterized by angiogenesis.Neoplasms may be benign such as hemangioma, glioma, or teratoma, ormalignant such as carcinoma, sarcoma, glioma, astrocytoma,neuroblastoma, or retinoblastoma.

The term “cancer tissue” refers to a tissue containing at least onecancer cell. Therefore, as cancer tissues contain cancer cells and bloodvessels, it refers to all cell types contributing to the formation of atumor mass containing cancer cells and endothelial cells. Herein, “tumormass” refers to a foci of tumor tissue. The term “tumor” is generallyused to mean a benign neoplasm or a malignant neoplasm.

For example, in several embodiments, cancer tissue-specific compoundsmay be compounds defined by qualitative properties of cancer tissuessuch as being present in cancer tissues but absent in non-cancertissues, or being absent in cancer tissues but present in non-cancertissues. In other embodiments, cancer tissue-specific compounds may becompounds defined by quantitative properties of cancer tissues such asbeing present in cancer tissues at a concentration different (forexample, higher concentration or lower concentration) from that innon-cancer tissues. For example, cancer tissue-specific compounds arepresent differentially at arbitrary concentrations. Generally, cancertissue-specific compounds can be present at a concentration increased byat least 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 100%, atleast 110%, at least 120%, at least 130%, at least 140%, at least 150%,at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, atleast 100-fold, at least 10³-fold, at least 10⁴-fold, at least 10⁵-fold,at least 10⁶-fold, or more, or up to infinity (i.e., when the compoundis absent in non-cancerous tissues). Alternatively, they can generallybe present at a concentration decreased by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 100% (i.e., absent). Preferably, cancertissue-specific compounds are differentially present at statisticallysignificant concentrations (that is, as determined using either Welch'st-test or Wilcoxon rank sum test, the p value is less than 0.05 and/orthe q value is less than 0.10). Examples of a non-limiting embodiment ofa cancer tissue-specific compound include compounds which are cancertissue-specific metabolites produced by metabolic activitiescharacteristic of cancer cells, immune cells, or stromal cells containedin cancer tissues, such as those described below (cancer tissue-specificmetabolites, cancer cell-specific metabolites, metabolites specific toimmune cells that infiltrated into cancer tissues, and cancer stromalcell-specific metabolites).

Cancer Tissue-Specific Metabolites

The term “metabolism” refers to chemical changes that take place inbiological tissues and includes “anabolism” and “catabolism”. Anabolismrefers to biosynthesis or accumulation of molecules, and catabolismrefers to degradation of molecules. “Metabolites” are intermediates orproducts that arise from metabolism. “Primary metabolites” refers tometabolites directly involved in the process of growth or proliferationof cells or organisms. “Secondary metabolites” refer to products thatare not directly involved in such process of growth or proliferation,and are products such as pigments or antibiotics that are produced as aresult of metabolism which biosynthesizs substances that are notdirectly involved in biological phenomena common to cells and organisms.The metabolites may be metabolites of “biopolymers”, or they may bemetabolites of “small molecules”. “Biopolymers” are polymers comprisingone or more types of repeating units. Biopolymers are generally found inbiological systems, and examples include cells forming the organism andintercellular matrices that adhere to them, molecules having a molecularweight of approximately 5000 or more which form structures such asinterstitial matrices, particularly polysaccharides (carbohydrates andsuch), peptides (this term is used so as to include polypeptides andproteins), and polynucleotides, and similarly their analogs such ascompounds composed of or including amino acid analogs or non-amino acidgroups. “Small molecules” refers to natural chemical substances otherthan “biopolymers” that exist in vivo. Suitable examples of anon-limiting embodiment of a cancer tissue-specific metabolite describedherein include cancer cell-specific small-molecule metabolites (EvaGottfried, Katrin Peter and Marina P. Kreutz, From Molecular to ModularTumor Therapy (2010) 3 (2), 111-132). In addition, metabolites that arehighly produced by immune cells that infiltrate into cancer tissues, andmetabolites that are highly produced by stromal cells that support thesurvival and/or growth of cancer cells (cancer stromal cells or cancerassociated stromal fibroblasts (CAF)) are also included. Infiltratingimmune cells are, for example, dendritic cells, inhibitory dendriticcells, inhibitory T cells, exhausted T cells, and myeloma derivedsuppressor cells (MDSC). Furthermore, metabolites of the presentinvention include compounds released from inside the cells to outsidethe cells when cells present in cancer tissues (cancer cells, immunecells, or stromal cells) die due to apoptosis, necrosis, or such.

To identify cancer cell-specific metabolites, metabolomic analysesfocused on metabolic profiling can be suitably used, in addition totranscriptome-level analyses (for example, Dhanasekaran et al. (Nature(2001) 412, 822-826), Lapointe et al. (Proc. Natl. Acad. Sci. U.S.A.(2004) 101, 811-816) or Perou et al. (Nature (2000) 406, 747-752)) andproteome-level analyses (for example, Ahram et al. (Mol. Carcinog.(2002) 33, 9-15), Hood et al. (Mol. Cell. Proteomics (2005) 4,1741-1753)). More specifically, to identify metabolites in test samples,metabolic profiling that uses high-pressure liquid chromatography(HPLC), nuclear magnetic resonance (NMR) (Brindle et al. (J. Mol.Recognit. (1997) 10, 182-187), mass spectrometry (Gates and Sweeley(Clin. Chem. (1978) 24, 1663-1673) (GC/MS and LC/MS)), and ELISA or suchindividually and/or in combination may be used appropriately.

These studies elucidated heterogeneity within the constituted tumorswhich results from changing the concentration gradient of growth factorsand metabolites (glucose, oxygen, or such) that enable cancer cellgrowth under low oxygen pressure conditions (Dang and Semenza (TrendsBiochem. Sci. (1999) 24, 68-72)). In these studies, cell line models arealso used to understand the change in energy utilization pathwaydepending on the different malignancy levels of tumors (Vizan et al.(Cancer Res. (2005) 65, 5512-5515)). Examples of a non-limitingembodiment of the technical components of the metabolomics platforminclude sample extraction, separation, detection, spectroscopicanalysis, data normalization, description of class-specific metabolites,pathway mapping, confirmation, and functional characterization ofcandidate metabolites described by Lawton et al. (Pharmacogenomics(2008) 9, 383). These methods enable identification of cancercell-specific metabolites in desired cancer tissues.

Examples of a non-limiting embodiment of cancer tissue-specificcompounds or cancer tissue-specific metabolites used in the presentinvention preferably include at least one compound selected from thecompounds below. At least one compound means that in addition to caseswhere the antigen-binding activity of a same antigen-binding domaindescribed below depends on one type of cancer tissue-specific compoundor metabolite, cases where it depends on several types of cancertissue-specific compounds or metabolites are included.

(1) Primary Metabolites of the Krebs Cycle or of the Glycolytic SystemSuch as Lactic Acid, Succinic Acid, and Citric Acid

Preferable examples of a non-limiting embodiment of a cancertissue-specific compound, particularly a cancer cell-specificmetabolite, used in the present invention include primary metabolitessuch as lactic acid, succinic acid, and citric acid, which are producedas a result of glucose metabolism, and are present at higherconcentrations in cancer tissues as compared to in the surroundingnon-cancerous tissues. The glycolytic system phenotype, which ischaracterized as an up-regulation of enzymes of the glycolytic system(Embden-Meyerhof pathway) such as pyruvate kinase, hexokinase, andlactic acid dehydrogenase (LDH), has been conventionally known to be acharacteristic of solid tumors as Warburg effect.

That is, in tumor cells, high expression of the pyruvate kinase isoformM2 which is necessary for anaerobic glycolysis, and not isoform Ml, isconsidered to be working advantageously for the growth of tumor cells invivo (Christofk et al. (Nature (2008) 452, 230-233). Pyruvic acidproduced by pyruvate kinase is subjected to feedback inhibition bylactic acid produced as a result of equilibrium reaction by lactic aciddehydrogenase (LDH) under anaerobic conditions. Since the feedbackinhibition causes promotion of respiration in mitochondria (Krebs cycle)and cell growth inhibition, up regulation of LDH, hexokinase, andglucose transporter (GLUT) is said to play an important role in theproliferation of cancer cells (Fantin et al. (Cancer Cell (2006) 9,425-434)). Glucose is metabolized by the glycolytic system, and thefinal metabolite lactic acid is transported together with protons to thetumor surrounding, and as a result, the pH of the tissues surroundingthe tumor is said to become acidic. Lactic acid, which is the finalproduct of the glycolytic pathway, as well as succinic acid and citricacid produced by promotion of respiration in mitochondria are known tobe accumulated in cancer tissues (Teresa et al. (Mol. Cancer (2009) 8,41-59)). Examples of a non-limiting embodiment of cancer tissue-specificcompounds, particularly cancer cell-specific metabolites, used in thepresent invention preferably include such primary metabolites such aslactic acid, succinic acid, and citric acid produced by metabolism bythe glycolytic pathway. Furthermore, succinic acid which is present athigh concentration in cells is known to leak out to the outside of thecells upon cell death (Nature Immunology, (2008) 9, 1261-1269).Therefore, succinic acid concentration is thought to be increased incancer tissues in which cell death occurs frequently.

(2) Amino Acids Such as Alanine, Glutamic Acid, and Aspartic Acid

Besides the above-mentioned glucose metabolism, the amino acidmetabolism is also known to be altered in tumor cells which requirecontinuous supply of essential amino acids and non-essential amino acidsthat are necessary for the biosynthesis of biopolymers under anaerobicconditions. Glutamine which contains two nitrogens in its side chainacts as a nitrogen transporter, and is an amino acid that is most widelydistributed in an organism. Tumor cells, in which the rate of glutamineuptake into cells is increased, is said to be functioning as a glutaminetrap. Such increase in the uptake of glutamine and activity ofconverting into glutamic acid and lactic acid is called“glutaminolysis”, and is considered to be a characteristic oftransformed (tumor) cells (Mazurek and Eigenbrodt (Anticancer Res.(2003) 23, 1149-1154); and Mazurek et al. (J. Cell. Physiol. (1999) 181,136-146)). As a result, cancer patients show an increase in glutamicacid concentration while showing a decrease in plasma glutamine level(Droge et al. (Immunobiology (1987) 174, 473-479)). Furthermore,correlation was observed between concentrations of ¹³C-labeled succinicacid, ¹³C-labeled alanine, ¹³C-labeled glutamic acid, and ¹³C-labeledcitric acid in studies on ¹³C-radiolabeled glucose metabolism in lungcancer tissues. Suitable examples of a non-limiting embodiment of cancertissue-specific compounds used in this invention include alanine,glutamic acid, and aspartic acid which accumulate at high concentrationsin cancer tissues through such glutaminolysis and the like.

(3) Amino Acid Metabolite Such as Kynurenine

Indolamine 2,3-dioxygenase (IDO) is a tryptophan-metabolizing enzymewhich is highly expressed in many cancers such as melanoma, coloncancer, and kidney cancer (Uyttenhove et al. (Nat. Med. (2003) 9,1269-127)); and it is known to have two isoforms (Lob et al. (CancerImmunol. Immunother. (2009) 58, 153-157)). IDO catalyzes the conversionof tryptophan to kynurenine (shown as Compound 1), and is the firstenzyme in the nicotinamide nucleotide (NAD) de novo pathway.Furthermore, in glioma which does not express IDO, kynurenine isproduced from tryptophan by tryptophan 2,3-dioxygenase (TDO) in theliver (Opitz et al. (Nature (2011) 478, 7368, 197-203)). IDO is alsoexpressed in dendritic cells infiltrated into cancer tissues, anddendritic cells also produce kynurenine (J. Immunol. (2008) 181,5396-5404). IDO is also expressed in myeloid-derived suppressor cells(MDSC) in cancer tissues, and MDSC also produces kynurenine (Yu et al.(J. Immunol. (2013) 190, 3783-3797)).

Kynurenine is known to suppress the same type of T cell response(Frumento et al. (J. Exp. Med. (2002) 196, 459-468); and a mechanism hasbeen suggested, in which tumor cells evade antitumor immune responsesthrough such inhibition, and proliferation of glioma cells is promotedthrough an autocrine proliferation mechanism in which kynurenine acts asan endogenous ligand for the aryl hydrocarbon receptor expressed ongliomas (Optiz et al. (mentioned above)). Kynurenine is converted toanthranilic acid (shown as Compound 2) by kynurenidase, and to3-hydroxykynurenine (shown as Compound 3) by kynurenine 3-hydroxylase.Anthranilic acid and 3-hydroxykynurenine are both converted to3-hydroxyanthranilic acid, the precursor of NAD.

Kynurenine is converted to kynurenic acid (shown as Compound 4) bykynurenine aminotransferase. Examples of a non-limiting embodiment ofcancer tissue-specific compounds, particularly cancer cell-specificmetabolites, used in the present invention preferably include such aminoacid metabolites such as kynurenine and its metabolites such asanthranilic acid, 3-hydroxykynurenine, and kynurenic acid.

(4) Arachidonic Acid Metabolites Such as Prostaglandin E2

Prostaglandin E2 (PGE2) (Compound 5) is an arachidonic acid metabolitecalled a prostanoid, which includes thromboxane and prostaglandinsynthesized by cyclooxygenase (COX)-1/2 (Warner and Mitchell (FASEB J.(2004) 18, 790-804)). PGE2 promotes the proliferation of colon cancercells and suppresses their apoptosis (Sheng et al. (Cancer Res. (1998)58, 362-366)). Cyclooxygenase expression is known to be altered in manycancer cells. More specifically, while COX-1 is expressed constitutivelyin almost all tissues, COX-2 has been found to be mainly induced bycertain types of inflammatory cytokines and cancer genes in tumors(Warner and Mitchell (mentioned above)). In addition, COX-2overexpression has been reported to be related to bad prognosis forbreast cancer (Denkert et al. (Clin. Breast Cancer (2004) 4, 428-433)),and rapid disease progression for ovarian cancer (Denker et al. (Mod.Pathol. (2006) 19, 1261-1269)). Inhibitory T cells that have infiltratedinto cancer tissues also produce prostaglandin E2 (Curr. Med. Chem.(2011) 18, 5217-5223). Small molecules such as the arachidonic acidmetabolites prostaglandin and leukotriene are known to act as astimulating factor that regulates autocrine and/or paracrine growth ofcancer (Nat. Rev. Cancer (2012) 12 (11) 782-792). Examples of anon-limiting embodiment of cancer tissue-specific compounds used in thepresent invention, particularly cancer cell-specific metabolites andimmune cell-specific metabolites that have infiltrated into cancertissues, preferably include such arachidonic acid metabolites such asprostaglandin E2. Besides prostaglandin E2, production of thromboxane A2(TXA2) is enhanced in cancer tissues such as colorectal cancer tissues(J. Lab. Clin. Med. (1993) 122, 518-523), and thromboxane A2 can besuitably presented as a non-limiting embodiment of an arachidonic acidmetabolite of the present invention.

(5) Nucleosides Carrying a Purine Ring Structure Such as Adenosine,Adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), and AdenosineMonophosphate (AMP)

When cancer cells undergo cell death, a large amount of ATP in the cellis known to leak out to the outside of the cells. Therefore, the ATPconcentration is remarkably higher in cancer tissues than in normaltissues (PLoS One. (2008) 3, e2599). Multiple types of cells releaseadenine nucleotides in the form of ATP, ADP, and AMP. Metabolism takesplace through an extracellular enzyme on the cell surface such asextracellular 5′-nucleotidase (ecto-5′-nucleotidase) (CD73) (Resta andThompson (Immunol. Rev. (1998) 161, 95-109) and Sadej et al. (MelanomaRes. (2006) 16, 213-222)). Adenosine is a purine nucleoside that existsconstitutively at low concentration in the extracellular environment,but in hypoxic tissues found in solid cancers, a remarkable increase inthe extracellular adenosine concentration has been reported (Blay andHoskin (Cancer Res. (1997) 57, 2602-2605). CD73 is expressed on thesurface of immune cells and tumors (Kobie et al. (J. Immunol. (2006)177, 6780-6786)), and its activity has been found to be increased inbreast cancer (Canbolat et al. (Breast Cancer Res. Treat. (1996) 37,189-193)), stomach cancer (Durak et al. (Cancer Lett. (1994) 84,199-202)), pancreatic cancer (Flocke and Mannherz (Biochim. Biophys.Acta (1991) 1076, 273-281), and glioblastoma (Bardot et al. (Br. J.Cancer (1994) 70, 212-218)). It has been proposed that the accumulationof adenosine in cancer tissues may be caused by an increase in theintracellular adenosine production through dephosphorylation of AMP by5′-nucleotidase in the cytoplasm (Headrick and Willis (Biochem. J.(1989) 261, 541-550)). Furthermore, inhibitory T cells and such thathave infiltrated into cancer tissues also express ATPase and produceadenosine (Proc. Natl. Acad. Sci. (2006) 103 (35), 13132-13137; Curr.Med. Chem. (2011) 18, 5217-5223). The produced adenosine is consideredto be rendering the cancer tissue an immunosuppressive environmentthrough adenosine receptors such as the A2A receptor (Curr. Med. Chem.(2011), 18, 5217-23). Examples of a non-limiting embodiment of thecancer tissue-specific compound used in the present invention preferablyinclude ATP, ADP, AMP, and adenosine which accumulate at highconcentration in cancer tissues through such metabolism of purinenucleotides such as ATP. Furthermore, since adenosine is degraded toinosine by adenosine deaminase, inosine accumulates at highconcentration.

(6) Uric Acid

Uric acid is a product of the metabolic pathway of purine nucleosides invivo, and is released to the outside of cells such as the interstitialspace and blood. In recent years, it has been found to be released fromdead cells that are present at sites of lesions such as cancer tissues(Nat. Med. (2007) 13, 851-856). Examples of a non-limiting embodiment ofcancer tissue-specific compounds used in the present inventionpreferably include such uric acid which accumulates at highconcentration in cancer tissues due to metabolism of purine nucleotidessuch as ATP.

(7) 1-Methyl Nicotinamide

The enzyme nicotinamide N-methyl transferase is known to be highlyexpressed in several human cancer tissues. When this enzyme produces thestable metabolite 1-methylnicotinamide from nicotinamide, the methylgroup of S-adenosylmethionine (SAM) which serves as a methyl donor isconsumed; therefore, the high expression of nicotinamideN-methyltransferase has been suggested to contribute to tumorigenesisthrough a mechanism that impairs the DNA methylation abilityaccompanying a decrease in the SAM concentration in cancer cells(Ulanovskaya et al. (Nat. Chem. Biol. (2013) 9 (5) 300-306)). The stablemetabolite of this enzyme, 1-methylnicotinamide is known to be secretedto the outside of cancer cells (Yamada et al. (J. Nutr. Sci. Vitaminol.(2010) 56, 83-86)), and preferable examples of a non-limiting embodimentof cancer tissue-specific compounds used in the present inventioninclude 1-methylnicotinamide and such which accumulate at highconcentration in cancer tissues through nicotinamide metabolism.

Inflammatory Tissue-Specific Compounds

The term “compound specific to inflammatory tissue (inflammatorytissue-specific compound)” as used herein refers to a compound that ispresent differentially in inflammatory tissues as compared tonon-inflammatory tissues. Herein, suitable examples of “inflammatorytissues” include:

joints with rheumatoid arthritis or osteoarthritis;lungs (alveoli) with bronchial asthma or COPD;digestive organs of inflammatory bowel disease, Crohn's disease, orulcerative colitis;fibrotic tissues of fibrosis of the liver, kidney, or lung;tissues undergoing rejection reaction in organ transplantation;blood vessels and heart (myocardium) in arteriosclerosis or heartfailure;visceral fat in metabolic syndrome;skin tissues in atopic detrmatitis or other dermatitis; andspinal nerves in disk herniation or chronic low back pain.

Inflammatory Tissue-Specific Metabolites

“Inflammatory tissue-specific metabolite” refers to metabolites highlyproduced by immune cells that have infiltrated into inflammatorytissues, and metabolites highly produced by specifically normal cellsthat have been damaged in inflammatory tissues. Examples of infiltratingimmune cells include effector T cells, mature dendritic cells,neutrophils, granule cells (mast cells), and basophils. Furthermore,metabolites in the present invention include compounds that are releasedfrom inside the cells to the outside of the cells when the cells thatare present in inflammatory tissues (immune cells and normal cells) dieby apoptosis, necrosis, or such.

Examples of a non-limiting embodiment of the inflammatorytissue-specific compounds or inflammatory tissue-specific metabolitesused in the present invention preferably include at least one compoundselected from the compounds below. At least one compound means includingcases where the antigen-binding activity of a same antigen-bindingdomain described below depends on one type of inflammatorytissue-specific compound or metabolite, as well as cases where itdepends on several types of inflammatory tissue-specific compounds ormetabolites.

(1) Arachidonic Acid Metabolites Such as Prostaglandin E2

The PGE2 concentration has been known to be high in rheumatoid arthritisand osteoarthritis (Eur. J. Clin. Pharmacol. (1994) 46, 3-7.; Clin. Exp.Rheumatol. (1999) 17, 151-160; Am. J. Vet. Res. (2004) 65, 1269-1275).Examples of a non-limiting embodiment of inflamatory tissue-specificcompounds, particularly inflammatory tissue-specific metabolites andmetabolites specific to immune cells that infiltrate into inflammatorytissues used in the present invention preferably include sucharachidonic acid metabolites such as prostaglandin E2.

(2) Nucleosides Carrying a Purine Ring Structure Such as Adenosine,Adenosine Triphosphate (ATP), Adenosine Diphosphate (ADP), and AdenosineMonophosphate (AMP)

ATP concentration is known to be high in pulmonary alveoli whereinflammation caused by bronchial asthma is taking place (Nat. Med.(2007) 13, 913-919). ATP concentration is also known to be high inpulmonary alveoli where inflammation caused by COPD is taking place (Am.J. Respir. Crit. Care Med. (2010) 181, 928-934). Furthermore, adenosineconcentration has been observed to be high in the joint fluid ofrheumatoid arthritis patients (Journal of Pharmaceutical and BiomedicalAnalysis (2004) 36, 877-882). Furthermore, ATP concentration is known tobe high in tissues where a rejection reaction is taking place due toGVHD (Nat. Med. (2010) 16, 1434-1438). Adenosine concentration is knownto be enhanced in fibrotic tissues of the liver, kidney, and lung (FASEBJ. (2008) 22, 2263-2272; J. Immunol. (2006) 176, 4449-4458; J. Am. Soc.Nephrol. (2011) 22 (5), 890-901; PLoS ONE J. (2010) 5 (2), e9242).Furthermore, ATP concentration has been observed to be increased infibrotic tissues of pulmonary fibrosis patients (Am. J. Respir. Crit.Care Med. (2010) 182, 774-783). Examples of a non-limiting embodiment ofan inflammatory tissue-specific compound used in the present inventionsuitably include ATP, ADP, AMP, adenosine and such which accumulate athigh concentration in inflammatory tissues by metabolism of such purinenucleotides such as ATP. In addition, inosine accumulates at a highconcentration due to degradation of adenosine by adenosine deaminase toproduce inosine.

(3) Uric Acid

Uric acid is a product of the metabolic pathway of purine nucleosides invivo, and is released to the outside of cells such as the interstitialspace and blood. In recent years, uric acid released from cellsundergoing necrosis has been found to promote inflammatory response (J.Clin. Invest. (2010) 120 (6), 1939-1949). Examples of a non-limitingembodiment of inflammatory tissue-specific compounds to be used in thepresent invention suitably include such uric acid which accumulates athigh concentration in inflammatory tissues due to metabolism of purinenucleotides such as ATP.

Antigen-Binding Domain

Herein, an “antigen-binding domain” may be of any structure as long asit binds to an antigen of interest. Such domains preferably include, forexample:

antibody heavy-chain and light-chain variable regions;a module of about 35 amino acids called A domain which is contained inthe in vivo cell membrane protein Avimer (International Publication No.WO 2004/044011, International Publication No. WO 2005/040229);Adnectin containing the 1 OFn3 domain which binds to the protein moietyof fibronectin, a glycoprotein expressed on cell membrane (InternationalPublication No. WO 2002/032925);Affibody which is composed of a 58-amino acid three-helix bundle basedon the scaffold of the IgG-binding domain of Protein A (InternationalPublication No. WO 1995/001937);Designed Ankyrin Repeat proteins (DARPins) which are a region exposed onthe molecular surface of ankyrin repeats (AR) having a structure inwhich a subunit consisting of a turn comprising 33 amino acid residues,two antiparallel helices, and a loop is repeatedly stacked(International Publication No. WO 2002/020565);Anticalins and such, which are domains consisting of four loops thatsupport one side of a barrel structure composed of eight circularlyarranged antiparallel strands that are highly conserved among lipocalinmolecules such as neutrophil gelatinase-associated lipocalin (NGAL)(International Publication No. WO 2003/029462); andthe concave region formed by the parallel-sheet structure inside thehorseshoe-shaped structure constituted by stacked repeats of theleucine-rich-repeat (LRR) module of the variable lymphocyte receptor(VLR) which does not have the immunoglobulin structure and is used inthe system of acquired immunity in jawless vertebrate such as lamperyand hagfish (International Publication No. WO 2008/016854). Preferredantigen-binding domains of the present invention include, for example,those having antibody heavy-chain and light-chain variable regions.Preferred examples of antigen-binding domains include “single chain Fv(scFv)”, “single chain antibody”, “Fv”, “single chain Fv 2 (scFv2)”,“Fab”, and “F(ab′)2”.

The antigen-binding domains of antigen-binding molecules of the presentinvention can bind to an identical epitope. Such identical epitope canbe present, for example, in a protein comprising the amino acid sequenceof SEQ ID NO: 1. Alternatively, each of the antigen-binding domains ofantigen-binding molecules of the present invention can bind to adifferent epitope. Herein, the different epitope can be present in, forexample, a protein comprising the amino acid sequence of SEQ ID NO: 1.

Specificity

“Specific” means that one of the molecules that specifically bind doesnot substantially bind to molecules other than the single or pluralityof partner molecules it binds to. Furthermore, “specific” is also usedwhen an antigen-binding domain is specific to a particular epitope amongmultiple epitopes in an antigen. When an epitope bound by anantigen-binding domain is contained in multiple different antigens,antigen-binding molecules containing the antigen-binding domain can bindto various antigens that have the epitope. Here, “does not substantiallybind” is determined according to the method described in theabove-mentioned section on binding activity, and refers to the bindingactivity of a molecule that specifically binds to a molecule other thanthe partner molecule, where the binding activity is not more than 80%,normally not more than 50%, preferably not more than 30%, orparticularly preferably not more than 15% of the binding activity to itspartner molecule.

Cytotoxic Activity

In a non-limiting embodiment, the present invention providesantigen-binding molecules that comprise an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of acancer-tissue specific compound, and which have cytotoxic activityagainst cells expressing a membrane-type molecule on their cellmembrane; and pharmaceutical compositions comprising theseantigen-binding molecules as an active ingredient. In the presentinvention, cytotoxic activity includes, for example, antibody-dependentcell-mediated cytotoxicity (ADCC) activity, complement-dependentcytotoxicity (CDC) activity, and cytotoxic activity by T cells. In thepresent invention, CDC activity refers to cytotoxic activity by thecomplement system. On the other hand, ADCC activity refers to theactivity of immune cells to damage target cells when the immune cellsand such bind to the Fc region of antigen-binding molecules comprisingan antigen-binding domain that binds to a membrane-type moleculeexpressed on the cell membrane of target cells via an Fcγ receptorexpressed on the immune cells. Whether an antigen-binding molecule ofinterest has an ADCC activity or whether it has a CDC activity can bedetermined using known methods (for example, Current Protocols inImmunology, Chapter 7. Immunologic studies in humans, Editor, Coligan etal., (1993)).

Specifically, effector cells, complement solution, and target cells arefirst prepared.

(1) Preparation of Effector Cells

Spleen is removed from a CBA/N mouse or the like, and spleen cells aredispersed in an RPMI1640 medium (Invitrogen). After the cells are washedin the same medium containing 10% fetal bovine serum (FBS, HyClone),effector cells are prepared by adjusting the spleen cell concentrationto 5×10⁶/mL.

(2) Preparation of Complement Solution

Baby Rabbit Complement (CEDARLANE) is diluted 10-fold in a culturemedium (Invitrogen) containing 10% FBS to prepare a complement solution.

(3) Preparation of Target Cells

The target cells can be radioactively labeled by culturing cellsexpressing the antigen with 0.2 mCi of ⁵¹Cr-sodium chromate-(GEHealthcare Bio-Sciences) in a DMEM medium containing 10% FBS for onehour at 37° C. After radioactive labeling, cells are washed three timesin an RPMI1640 medium containing 10% FBS, and the target cells can beprepared by adjusting the cell concentration to 2×10⁵/mL.

ADCC activity or CDC activity can be measured by the method describedbelow. In the case of ADCC activity measurement, 50 μL each of thetarget cell and antigen-binding molecule are added to a 96-well U-bottomplate (Becton Dickinson), and allowed to react for 15 minutes at roomtemperature. Then, 100 μL of effector cells are added to the plate andthis plate is placed in a carbon dioxide incubator for four hours. Thefinal concentration of the antigen-binding molecule may be set, forexample, to 0 μg/mL or 10 μg/mL. After incubation, 100 μL of thesupernatant is collected from each well, and the radioactivity ismeasured with a gamma counter (COBRAII AUTO-GAMMA, MODEL D5005, PackardInstrument Company). The cytotoxic activity (%) can be calculated usingthe measured values according to the equation: (A−C)/(B−C)×100. Arepresents the radioactivity (cpm) in each sample, B represents theradioactivity (cpm) in a sample to which 1% NP-40 (Nacalai Tesque) hasbeen added, and C represents the radioactivity (cpm) of a samplecontaining the target cells alone.

Meanwhile, in the case of CDC activity measurement, 50 μL of target celland 50 μL of an antigen-binding molecule are added to a 96-wellflat-bottomed plate (Becton Dickinson), and allowed to react for 15minutes on ice. Then, 100 μL of a complement solution is added to theplate, and this plate is placed in a carbon dioxide incubator for fourhours. The final concentration of the antigen-binding molecule may beset, for example, to 0 μg/mL or 3 μg/mL. After incubation, 100 μL ofsupernatant is collected from each well, and the radioactivity ismeasured with a gamma counter. The cytotoxic activity can be calculatedin the same way as in the determination of ADCC activity.

The later-described modified antigen-binding molecules to whichcytotoxic substances such as chemotherapeutic agents, toxic peptides, orradioactive chemical substances have been ligated can also be suitablyused as the antigen-binding molecules of the present invention havingcytotoxic activity. Such modified antigen-binding molecules (hereinafterreferred to as “antigen-binding molecule-drug conjugate”) can beobtained by chemically modifying the obtained antigen-binding molecules.Methods that have been already established in the field of antibody-drugconjugates and such may be used appropriately as a method for modifyingantigen-binding molecules. Furthermore, a modified antigen-bindingmolecule with a linked toxic peptide can be obtained by expressing in anappropriate host cell a fusion gene produced by linking a gene encodingthe toxic peptide in frame with a gene encoding an antigen-bindingmolecule of the present invention, and then isolating the molecule fromthe culture solution of the cells.

Neutralizing Activity

The present invention provides in a non-limiting embodiment apharmaceutical composition that induces an immune response, comprisingas an active ingredient an antigen-binding molecule that contains anantigen-binding domain whose antigen-binding activity varies dependingon the concentration of a cancer tissue-specific compound and has aneutralizing activity against a membrane-type molecule. In anothernon-limiting embodiment, the present invention provides a pharmaceuticalcomposition that induces an immune response, comprising as an activeingredient an antigen-binding molecule that contains an antigen-bindingdomain whose antigen-binding activity varies depending on theconcentration of a cancer tissue-specific compound and has aneutralizing activity against a membrane-type molecule in addition to acytotoxic activity against cells expressing the membrane-type moleculeon their cell membrane. Generally, a neutralizing activity refers to anactivity of inhibiting the biological activity of a ligand which has abiological activity towards cells, such as viruses and toxins. Thus, asubstance having a neutralizing activity refers to a substance thatbinds to a ligand or a receptor to which the ligand binds and inhibitsthe binding between the ligand and the receptor. A receptor whosebinding to the ligand has been blocked by the neutralizing activity willnot be able to exhibit the biological activity through the receptor.When the antigen-binding molecule is an antibody, the antibody havingsuch a neutralizing activity is generally called a neutralizingantibody. The neutralizing activity of a test substance may be measuredby comparing the biological activities in the presence of a ligandbetween conditions when the test substance is present or absent.

A suitable example of a major ligand for the IL-6 receptor is IL-6,which is shown in SEQ ID NO: 27. The IL-6 receptor, which is an I-typemembrane protein whose amino terminus forms the extracellular domain,forms a hetero-tetramer with the gp130 receptor which was induced byIL-6 to dimerize (Heinrich et al. (Biochem. J. (1998) 334, 297-314)).Formation of the heterotetramer activates Jak associated with the gp130receptor. Jak carries out autophosphorylation and receptorphosphorylation. The phosphorylation sites of the receptor and of Jakserve as binding sites for molecules belonging to the Stat family havingSH2 such as Stat3, and for the MAP kinases, PI3/Akt, and other proteinsand adapters having SH2. Next, Stat that bound to the gp130 receptor isphosphorylated by Jak. The phosphorylated Stat dimerizes andtranslocates to the nucleus, and regulates transcription of targetgenes. Jak and Stat can also be involved in the signaling cascadethrough receptors of other classes. A deregulated IL-6 signaling cascadeis observed in inflammation and pathological conditions of autoimmunediseases, and cancers such as prostate cancer and multiple myeloma.Stat3 which may act as an oncogene is constitutively activated in manycancers. In prostate cancer and multiple myeloma, there is a crosstalkbetween the signaling cascade from the IL-6 receptor and the signalingcascade from members of the epidermal growth factor receptor (EGFR)family (Ishikawa et al. (J. Clin. Exp. Hematopathol. (2006) 46 (2),55-66)).

Such intracellular signaling cascades are different for each cell type;therefore, an appropriate target molecule can be set according to eachof the target cells of interest, and the target molecule is not limitedto the above-mentioned factors. The neutralization activity can beevaluated by measuring the in vivo signal activation. Furthermore,activation of in vivo signals can also be detected by using as anindicator the transcription-inducing action on a target gene that existsdownstream of the in vivo signaling cascade. A change in thetranscription activity of a target gene can be detected by the principleof a reporter assay. Specifically, a reporter gene such as the greenfluorescence protein (GFP) or luciferase is placed downstream of atranscription factor or a promoter region of the target gene; and achange in transcription activity can be measured in terms of reporteractivity by measuring the reporter activity. Commercially available kitsfor measuring in vivo signal activation can be suitably used (forexample, the Mercury Pathway Profiling Luciferase System (Clontech)).

Furthermore, as a method for measuring the neutralization activity on areceptor ligand in the EGF receptor family and such which acts on asignaling cascade that typically works toward enhancing cellproliferation, neutralization activity of an antigen-binding moleculecan be evaluated by measuring the proliferation activity of the targetcells. For example, the following method is suitably used as a methodfor measuring or evaluating inhibitory effects based on theneutralization activity of an anti-HB-EGF antibody against theproliferation of cells whose proliferation is promoted by EGF familygrowth factors such as HB-EGF. As a method for evaluating or measuringthe activity of inhibiting cell proliferation in a test tube, a methodthat measures the incorporation by living cells of [³H]-labeledthymidine added to the culture medium as an index of the DNA replicationability is used. As a more convenient method, a dye exclusion methodthat measures under a microscope the ability of a cell to release a dyesuch as trypan blue to the outside of the cell, or the MTT method isused. The latter makes use of the ability of living cells to convert3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT),which is a tetrazolium salt, to a blue formazan product. Morespecifically, a test antibody is added along with a ligand to theculture solution of a test cell; and after a certain period of time haselapsed, an MTT solution is added to the culture, and this is left tostand for a certain amount of time to let the cell incorporate MTT. As aresult, MTT which is a yellow compound is converted to a blue compoundby succinate dehydrogenase in the mitochondria of the cell. After thisblue product is dissolved for coloration, its absorbance is measured andused as an indicator of the number of viable cells. Besides MTT,reagents such as MTS, XTT, WST-1, and WST-8 are also commerciallyavailable (Nacalai Tesque, and such), and can be suitably used. Formeasurement of the activity, a binding antibody that has the sameisotype as the anti-HB-EGF antibody but does not have the cellproliferation-inhibiting activity can be used as a control antibody inthe same manner as the anti-HB-EGF antibody, and the anti-HB-EGFantibody is judged to have the activity when it shows a stronger cellproliferation-inhibiting activity than the control antibody.

As cells for evaluating activity, for example, cells showingHB-EGF-promoted proliferation such as the RMG-1 cell line which is anovarian cancer cell line may be suitably used; and mouse Ba/F3 cellstransformed with a vector in which a gene encoding hEGFR/mG-CSFR, whichis a fusion protein of the extracellular domain of human EGFR fused inframe with the intracellular domain of the mouse G-CSF receptor, islinked so as to allow expression, may also be suitably used. This way,those skilled in the art may appropriately select cells for evaluatingactivity to measure the cell proliferation activity mentioned above.

Antibody

Herein, “antibody” refers to a natural immunoglobulin or animmunoglobulin produced by partial or complete synthesis. Antibodies canbe isolated from natural sources such as naturally-occurring plasma andserum, or culture supernatants of antibody-producing hybridomas.Alternatively, antibodies can be partially or completely synthesizedusing techniques such as genetic recombination. Preferred antibodiesinclude, for example, antibodies of an immunoglobulin isotype orsubclass belonging thereto. Known human immunoglobulins includeantibodies of the following nine classes (isotypes): IgG1, IgG2, IgG3,IgG4, IgA1, IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies ofthe present invention include IgG1, IgG2, IgG3, and IgG4. A number ofallotype sequences of human IgG1, human IgG2, human IgG3, and human IgG4constant regions due to gene polymorphisms are described in “Sequencesof proteins of immunological interest”, NIH Publication No. 91-3242. Anyof such sequences may be used in the present invention. In particular,for the human IgG1sequence, the amino acid sequence at positions 356 to358 as indicated by EU numbering may be DEL or EEM. Several allotypesequences due to genetic polymorphisms have been described in “Sequencesof proteins of immunological interest”, NIH Publication No. 91-3242 forthe human Igκ (Kappa) constant region and human Igλ (Lambda) constantregion, and any of the sequences may be used in the present invention.

Methods for producing an antibody with desired binding activity areknown to those skilled in the art. Below is an example that describes amethod for producing an antibody that binds to IL-6R (anti-IL-6Rantibody). Antibodies that bind to an antigen other than IL-6R can alsobe produced according to the example described below.

Anti-IL-6R antibodies can be obtained as polyclonal or monoclonalantibodies using known methods. The anti-IL-6R antibodies preferablyproduced are monoclonal antibodies derived from mammals. Suchmammal-derived monoclonal antibodies include antibodies produced byhybridomas or host cells transformed with an expression vector carryingan antibody gene by genetic engineering techniques. “Humanizedantibodies” or “chimeric antibodies” are included in the monoclonalantibodies of the present invention.

Monoclonal antibody-producing hybridomas can be produced using knowntechniques, for example, as described below. Specifically, mammals areimmunized by conventional immunization methods using an IL-6R protein asa sensitizing antigen. Resulting immune cells are fused with knownparental cells by conventional cell fusion methods. Then, hybridomasproducing an anti-IL-6R antibody can be selected by screening formonoclonal antibody-producing cells using conventional screeningmethods.

Specifically, monoclonal antibodies are prepared as mentioned below.First, the IL-6R gene whose nucleotide sequence is disclosed in SEQ IDNO: 2 can be expressed to produce an IL-6R protein shown in SEQ ID NO:1, which will be used as a sensitizing antigen for antibody preparation.That is, a gene sequence encoding IL-6R is inserted into a knownexpression vector, and appropriate host cells are transformed with thisvector. The desired human IL-6R protein is purified from the host cellsor their culture supernatants by known methods. In order to obtainsoluble IL-6R from culture supernatants, for example, a proteinconsisting of the amino acids at positions 1 to 357 in the IL-6Rpolypeptide sequence of SEQ ID NO: 1, such as described in Mullberg etal. (J. Immunol. (1994) 152 (10), 4958-4968), is expressed as a solubleIL-6R, instead of the IL-6R protein of SEQ ID NO: 1. Purified naturalIL-6R protein can also be used as a sensitizing antigen.

The purified IL-6R protein can be used as a sensitizing antigen forimmunization of mammals. A partial IL-6R peptide may also be used as asensitizing antigen. In this case, a partial peptide can be prepared bychemical synthesis based on the amino acid sequence of human IL-6R, orby inserting a partial IL-6R gene into an expression vector forexpression. Alternatively, a partial peptide can be produced bydegrading an IL-6R protein with a protease. The length and region of thepartial IL-6R peptide are not limited to particular embodiments. Apreferred region can be arbitrarily selected from the amino acidsequence at amino acid positions 20 to 357 in the amino acid sequence ofSEQ ID NO: 1. The number of amino acids forming a peptide to be used asa sensitizing antigen is preferably at least five or more, six or more,or seven or more. More specifically, a peptide of 8 to 50 residues, morepreferably 10 to 30 residues can be used as a sensitizing antigen.

For sensitizing antigen, alternatively it is possible to use a fusionprotein prepared by fusing a desired partial polypeptide or peptide ofthe IL-6R protein with a different polypeptide. For example, antibody Fcfragments and peptide tags are preferably used to produce fusionproteins to be used as sensitizing antigens. Vectors for expression ofsuch fusion proteins can be constructed by fusing in frame genesencoding two or more desired polypeptide fragments and inserting thefusion gene into an expression vector as described above. Methods forproducing fusion proteins are described in Molecular Cloning 2nd ed.(Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) ColdSpring Harbor Lab. Press). Methods for preparing IL-6R to be used as asensitizing antigen, and immunization methods using IL-6R arespecifically described in WO 2003/000883, WO 2004/022754, WO2006/006693, and such.

There is no particular limitation on the mammals to be immunized withthe sensitizing antigen. However, it is preferable to select the mammalsby considering their compatibility with the parent cells to be used forcell fusion. In general, rodents such as mice, rats, and hamsters,rabbits, and monkeys are preferably used.

The above animals are immunized with a sensitizing antigen by knownmethods. Generally performed immunization methods include, for example,intraperitoneal or subcutaneous injection administration of asensitizing antigen into mammals. Specifically, a sensitizing antigen isappropriately diluted with PBS (Phosphate-Buffered Saline),physiological saline, or the like. If desired, a conventional adjuvantsuch as Freund's complete adjuvant is mixed with the antigen, and themixture is emulsified. Then, the sensitizing antigen is administered toa mammal several times at 4- to 21-day intervals. Appropriate carriersmay be used in immunization with the sensitizing antigen. In particular,when a low-molecular-weight partial peptide is used as the sensitizingantigen, it is sometimes desirable to couple the sensitizing antigenpeptide to a carrier protein such as albumin or keyhole limpethemocyanin for immunization.

Alternatively, hybridomas producing a desired antibody can be preparedusing DNA immunization as mentioned below. DNA immunization is animmunization method that confers immunostimulation by expressing asensitizing antigen in an animal immunized as a result of administeringa vector DNA constructed to allow expression of an antigenprotein-encoding gene in the animal. As compared to conventionalimmunization methods in which a protein antigen is administered toanimals to be immunized, DNA immunization is expected to be superior inthat:

-   -   immunostimulation can be provided while retaining the structure        of a membrane protein such as IL-6R; and    -   there is no need to purify the antigen for immunization.

In order to prepare a monoclonal antibody of the present invention usingDNA immunization, first, a DNA expressing an IL-6R protein isadministered to an animal to be immunized. The IL-6R-encoding DNA can besynthesized by known methods such as PCR. The obtained DNA is insertedinto an appropriate expression vector, and then this is administered toan animal to be immunized. Preferably used expression vectors include,for example, commercially-available expression vectors such as pcDNA3.1.Vectors can be administered to an organism using conventional methods.For example, DNA immunization is performed by using a gene gun tointroduce expression vector-coated gold particles into cells in the bodyof an animal to be immunized. Antibodies that recognized IL-6R can alsobe produced by the methods described in WO 2003/104453.

After immunizing a mammal as described above, an increase in the titerof an IL-6R-binding antibody is confirmed in the serum. Then, immunecells are collected from the mammal, and then subjected to cell fusion.In particular, splenocytes are preferably used as immune cells.

A mammalian myeloma cell is used as a cell to be fused with theabove-mentioned immune cells. The myeloma cells preferably comprise asuitable selection marker for screening. A selection marker conferscharacteristics to cells for their survival (or death) under a specificculture condition. Hypoxanthine-guanine phosphoribosyltransferasedeficiency (hereinafter abbreviated as HGPRT deficiency) and thymidinekinase deficiency (hereinafter abbreviated as TK deficiency) are knownas selection markers. Cells with HGPRT or TK deficiency havehypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviatedas HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in a HATselection medium, and are thus killed. However, when the cells are fusedwith normal cells, they can continue DNA synthesis using the salvagepathway of the normal cells, and therefore they can grow even in the HATselection medium.

HGPRT-deficient and TK-deficient cells can be selected in a mediumcontaining 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG),or 5′-bromodeoxyuridine, respectively. Normal cells are killed becausethey incorporate these pyrimidine analogs into their DNA. Meanwhile,cells that are deficient in these enzymes can survive in the selectionmedium, since they cannot incorporate these pyrimidine analogs. Inaddition, a selection marker referred to as G418 resistance provided bythe neomycin-resistant gene confers resistance to 2-deoxystreptamineantibiotics (gentamycin analogs). Various types of myeloma cells thatare suitable for cell fusion are known.

For example, myeloma cells including the following cells can bepreferably used: P3(P3x63Ag8.653) (J. Immunol. (1979) 123 (4),1548-1550);

P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978)81,1-7); NS-1 (C. Eur. J. Immunol. (1976)6 (7), 511-519);

MPC-11 (Cell (1976) 8 (3), 405-415); SP2/0 (Nature (1978) 276 (5685),269-270);

FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);S194/5.XXO.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);

R210 (Nature (1979) 277 (5692), 131-133), etc.

Cell fusions between the immunocytes and myeloma cells are essentiallycarried out using known methods, for example, a method by Kohler andMilstein et al. (Methods Enzymol. (1981) 73: 3-46).

More specifically, cell fusion can be carried out, for example, in aconventional culture medium in the presence of a cell fusion-promotingagent. The fusion-promoting agents include, for example, polyethyleneglycol (PEG) and Sendai virus (HVJ). If required, an auxiliary substancesuch as dimethyl sulfoxide is also added to improve fusion efficiency.

The ratio of immune cells to myeloma cells may be determined at one'sown discretion, preferably, for example, one myeloma cell for every oneto ten immunocytes. Culture media to be used for cell fusions include,for example, media that are suitable for the growth of myeloma celllines, such as RPMI1640 medium and MEM medium, and other conventionalculture medium used for this type of cell culture. In addition, serumsupplements such as fetal calf serum (FCS) may be preferably added tothe culture medium.

For cell fusion, predetermined amounts of the above immune cells andmyeloma cells are mixed well in the above culture medium. Then, a PEGsolution (for example, the average molecular weight is about 1,000 to6,000) prewarmed to about 37° C. is added thereto at a concentration ofgenerally 30% to 60% (w/v). This is gently mixed to produce desiredfusion cells (hybridomas). Then, an appropriate culture medium mentionedabove is gradually added to the cells, and this is repeatedlycentrifuged to remove the supernatant. Thus, cell fusion agents and suchwhich are unfavorable to hybridoma growth can be removed.

The hybridomas thus obtained can be selected by culture using aconventional selective medium, for example, HAT medium (a culture mediumcontaining hypoxanthine, aminopterin, and thymidine). Cells other thanthe desired hybridomas (non-fused cells) can be killed by continuingculture in the above HAT medium for a sufficient period of time.Typically, the period is several days to several weeks. Then, hybridomasproducing the desired antibody are screened and singly cloned byconventional limiting dilution methods.

The hybridomas thus obtained can be selected using a selection mediumbased on the selection marker possessed by the myeloma used for cellfusion. For example, HGPRT- or TK-deficient cells can be selected byculture using the HAT medium (a culture medium containing hypoxanthine,aminopterin, and thymidine). Specifically, when HAT-sensitive myelomacells are used for cell fusion, cells successfully fused with normalcells can selectively proliferate in the HAT medium. Cells other thanthe desired hybridomas (non-fused cells) can be killed by continuingculture in the above HAT medium for a sufficient period of time.Specifically, desired hybridomas can be selected by culture forgenerally several days to several weeks. Then, hybridomas producing thedesired antibody are screened and singly cloned by conventional limitingdilution methods.

Desired antibodies can be preferably selected and singly cloned byscreening methods based on known antigen/antibody reaction. For example,an IL-6R-binding monoclonal antibody can bind to IL-6R expressed on thecell surface. Such a monoclonal antibody can be screened by fluorescenceactivated cell sorting (FACS). FACS is a system that assesses thebinding of an antibody to cell surface by analyzing cells contacted witha fluorescent antibody using laser beam, and measuring the fluorescenceemitted from individual cells.

To screen for hybridomas that produce a monoclonal antibody of thepresent invention by FACS, IL-6R-expressing cells are first prepared.Cells preferably used for screening are mammalian cells in which IL-6Ris forcedly expressed. As control, the activity of an antibody to bindto cell-surface IL-6R can be selectively detected using non-transformedmammalian cells as host cells. Specifically, hybridomas producing ananti-IL-6R monoclonal antibody can be isolated by selecting hybridomasthat produce an antibody which binds to cells forced to express IL-6R,but not to host cells.

Alternatively, the activity of an antibody to bind to immobilizedIL-6R-expressing cells can be assessed based on the principle of ELISA.For example, IL-6R-expressing cells are immobilized to the wells of anELISA plate. Culture supernatants of hybridomas are contacted with theimmobilized cells in the wells, and antibodies that bind to theimmobilized cells are detected. When the monoclonal antibodies arederived from mouse, antibodies bound to the cells can be detected usingan anti-mouse immunoglobulin antibody. Hybridomas producing a desiredantibody having the antigen-binding ability are selected by the abovescreening, and they can be cloned by a limiting dilution method or thelike.

Monoclonal antibody-producing hybridomas thus prepared can be passagedin a conventional culture medium, and stored in liquid nitrogen for along period.

The above hybridomas are cultured by a conventional method, and desiredmonoclonal antibodies can be prepared from the culture supernatants.Alternatively, the hybridomas are administered to and grown incompatible mammals, and monoclonal antibodies are prepared from theascites. The former method is suitable for preparing antibodies withhigh purity.

Antibodies encoded by antibody genes that are cloned fromantibody-producing cells such as the above hybridomas can also bepreferably used. A cloned antibody gene is inserted into an appropriatevector, and this is introduced into a host to express the antibodyencoded by the gene. Methods for isolating antibody genes, inserting thegenes into vectors, and transforming host cells have already beenestablished, for example, by Vandamme et al. (Eur. J. Biochem. (1990)192(3), 767-775). Methods for producing recombinant antibodies are alsoknown as described below.

For example, a cDNA encoding the variable region (V region) of ananti-IL-6R antibody is prepared from hybridoma cells expressing theanti-IL-6R antibody. For this purpose, total RNA is first extracted fromhybridomas. Methods used for extracting mRNAs from cells include, forexample:

-   -   the guanidine ultracentrifugation method (Biochemistry (1979)        18(24), 5294-5299), and    -   the AGPC method (Anal. Biochem. (1987) 162(1), 156-159)

Extracted mRNAs can be purified using the mRNA Purification Kit (GEHealthcare Bioscience) or such. Alternatively, kits for extracting totalmRNA directly from cells, such as the QuickPrep mRNA Purification Kit(GE Healthcare Bioscience), are also commercially available. mRNAs canbe prepared from hybridomas using such kits. cDNAs encoding the antibodyV region can be synthesized from the prepared mRNAs using a reversetranscriptase. cDNAs can be synthesized using the AMV ReverseTranscriptase First-strand cDNA Synthesis Kit (Seikagaku Co.) or such.Furthermore, the SMART RACE cDNA amplification kit (Clontech) and thePCR-based 5′-RACE method (Proc. Natl. Acad. Sci. U.S.A. (1988) 85(23),8998-9002; Nucleic Acids Res. (1989) 17(8), 2919-2932) can beappropriately used to synthesize and amplify cDNAs. In such a cDNAsynthesis process, appropriate restriction enzyme sites described belowmay be introduced into both ends of a cDNA.

The cDNA fragment of interest is purified from the resulting PCRproduct, and then this is ligated to a vector DNA. A recombinant vectoris thus constructed, and introduced into E. coli or such. After colonyselection, the desired recombinant vector can be prepared from thecolony-forming E. coli. Then, whether the recombinant vector has thecDNA nucleotide sequence of interest is tested by a known method such asthe dideoxy nucleotide chain termination method.

The 5′-RACE method which uses primers to amplify the variable regiongene is conveniently used for isolating the gene encoding the variableregion. First, a 5′-RACE cDNA library is constructed by cDNA synthesisusing RNAs extracted from hybridoma cells as a template. A commerciallyavailable kit such as the SMART RACE cDNA amplification kit isappropriately used to synthesize the 5′-RACE cDNA library.

The antibody gene is amplified by PCR using the prepared 5′-RACE cDNAlibrary as a template. Primers for amplifying the mouse antibody genecan be designed based on known antibody gene sequences. The nucleotidesequences of the primers vary depending on the immunoglobulin subclass.Therefore, it is preferable that the subclass is determined in advanceusing a commercially available kit such as the Iso Strip mousemonoclonal antibody isotyping kit (Roche Diagnostics).

Specifically, for example, primers that allow amplification of genesencoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains areused to isolate mouse IgG-encoding genes. In general, a primer thatanneals to a constant region site close to the variable region is usedas a 3′-side primer to amplify an IgG variable region gene. Meanwhile, aprimer attached to a 5′ RACE cDNA library construction kit is used as a5′-side primer.

PCR products thus amplified are used to reshape immunoglobulins composedof a combination of heavy and light chains. A desired antibody can beselected using the IL-6R-binding activity of a reshaped immunoglobulinas an indicator. For example, when the objective is to isolate anantibody against IL-6R, it is more preferred that the binding of theantibody to IL-6R is specific. An IL-6R-binding antibody can bescreened, for example, by the following steps:

(1) contacting an IL-6R-expressing cell with an antibody comprising theV region encoded by a cDNA isolated from a hybridoma;

(2) detecting the binding of the antibody to the IL-6R-expressing cell;and

(3) selecting an antibody that binds to the IL-6R-expressing cell.

Methods for detecting the binding of an antibody to IL-6R-expressingcells are known. Specifically, the binding of an antibody toIL-6R-expressing cells can be detected by the above-described techniquessuch as FACS. Immobilized samples of IL-6R-expressing cells areappropriately used to assess the binding activity of an antibody.

Preferred antibody screening methods that use the binding activity as anindicator also include panning methods using phage vectors. Screeningmethods using phage vectors are advantageous when the antibody genes areisolated from heavy-chain and light-chain subclass libraries from apolyclonal antibody-expressing cell population. Genes encoding theheavy-chain and light-chain variable regions can be linked by anappropriate linker sequence to form a single-chain Fv (scFv). Phagespresenting scFv on their surface can be produced by inserting a geneencoding scFv into a phage vector. The phages are contacted with anantigen of interest. Then, a DNA encoding scFv having the bindingactivity of interest can be isolated by collecting phages bound to theantigen. This process can be repeated as necessary to enrich scFv havingthe binding activity of interest.

After isolation of the cDNA encoding the V region of the anti-IL-6Rantibody of interest, the cDNA is digested with restriction enzymes thatrecognize the restriction sites introduced into both ends of the cDNA.Preferred restriction enzymes recognize and cleave a nucleotide sequencethat occurs in the nucleotide sequence of the antibody gene at a lowfrequency. Furthermore, a restriction site for an enzyme that produces asticky end is preferably introduced into a vector to insert asingle-copy digested fragment in the correct orientation. The cDNAencoding the V region of the anti-IL-6R antibody is digested asdescribed above, and this is inserted into an appropriate expressionvector to construct an antibody expression vector. In this case, if agene encoding the antibody constant region (C region) and a geneencoding the above V region are fused in-frame, a chimeric antibody isobtained. Herein, “chimeric antibody” means that the origin of theconstant region is different from that of the variable region. Thus, inaddition to mouse/human heterochimeric antibodies, human/humanallochimeric antibodies are included in the chimeric antibodies of thepresent invention. A chimeric antibody expression vector can beconstructed by inserting the above V region gene into an expressionvector that already has the constant region. Specifically, for example,a recognition sequence for a restriction enzyme that excises the above Vregion gene can be appropriately placed on the 5′ side of an expressionvector carrying a DNA encoding a desired antibody constant region. Achimeric antibody expression vector is constructed by fusing in framethe two genes digested with the same combination of restriction enzymes.

To produce an anti-IL-6R monoclonal antibody, antibody genes areinserted into an expression vector so that the genes are expressed underthe control of an expression regulatory region. The expressionregulatory region for antibody expression includes, for example,enhancers and promoters. Furthermore, an appropriate signal sequence maybe attached to the amino terminus so that the expressed antibody issecreted to the outside of cells. In the Examples below, a peptidehaving the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 3) isused as a signal sequence. Meanwhile, other appropriate signal sequencesmay be attached. The expressed polypeptide is cleaved at the carboxylterminus of the above sequence, and the resulting polypeptide issecreted to the outside of cells as a mature polypeptide. Then,appropriate host cells are transformed with the expression vector, andrecombinant cells expressing the anti-IL-6R antibody-encoding DNA areobtained.

DNAs encoding the antibody heavy chain (H chain) and light chain (Lchain) are separately inserted into different expression vectors toexpress the antibody gene. An antibody molecule having the H and Lchains can be expressed by co-transfecting the same host cell withvectors into which the H-chain and L-chain genes are respectivelyinserted. Alternatively, host cells can be transformed with a singleexpression vector into which DNAs encoding the H and L chains areinserted (see WO 1994/011523).

There are various known host cell/expression vector combinations forantibody preparation by introducing isolated antibody genes intoappropriate hosts. All of these expression systems are applicable toisolation of the antigen-binding domains of the present invention.Appropriate eukaryotic cells used as host cells include animal cells,plant cells, and fungal cells. Specifically, the animal cells include,for example, the following cells.

(1) mammalian cells: CHO (Chinese hamster ovary cell line), COS (Monkeykidney cell line), myeloma (Sp2/0, NSO, etc.), BHK (baby hamster kidneycell line), HeLa, Vero, HEK293 (human embryonic kidney cell line withsheared adenovirus (Ad)5 DNA), PER.C6 cell (human embryonic retinal cellline transformed with the Adenovirus Type 5 (Ad5) E1A and E1B genes) andsuch (Current Protocols in Protein Science (May, 2001, Unit 5.9, Table5.9.1));(2) amphibian cells: Xenopus oocytes, or such; and(3) insect cells: sf9, sf21, Tn5, or such.

In addition, as a plant cell, an antibody gene expression system usingcells derived from the Nicotiana genus such as Nicotiana tabacum isknown. Callus cultured cells can be appropriately used to transformplant cells.

Furthermore, the following cells can be used as fungal cells:

-   -   yeasts: the Saccharomyces genus such as Saccharomyces        serevisiae, and the Pichia genus such as Pichia pastoris; and    -   filamentous fungi: the Aspergillus genus such as Aspergillus        niger.

Furthermore, antibody gene expression systems that utilize prokaryoticcells are also known. For example, when using bacterial cells, E. colicells, Bacillus subtilis cells, and such can suitably be utilized in thepresent invention. Expression vectors carrying the antibody genes ofinterest are introduced into these cells by transfection. Thetransfected cells are cultured in vitro, and the desired antibody can beprepared from the culture of transformed cells.

In addition to the above-described host cells, transgenic animals canalso be used to produce a recombinant antibody. That is, the antibodycan be obtained from an animal into which the gene encoding the antibodyof interest is introduced. For example, the antibody gene can beconstructed as a fusion gene by inserting in frame into a gene thatencodes a protein produced specifically in milk. Goat β-casein or suchcan be used, for example, as the protein secreted in milk. DNA fragmentscontaining the fused gene inserted with the antibody gene is injectedinto a goat embryo, and then this embryo is introduced into a femalegoat. Desired antibodies can be obtained as a protein fused with themilk protein from milk produced by the transgenic goat born from theembryo-recipient goat (or progeny thereof). In addition, to increase thevolume of milk containing the desired antibody produced by thetransgenic goat, hormones can be administered to the transgenic goat asnecessary (Ebert, K. M. et al., Bio/Technology (1994) 12 (7), 699-702).

When an antigen-binding molecule described herein is administered tohuman, an antigen-binding domain derived from a genetically recombinantantibody that has been artificially altered to reduce the heterologousantigenicity against human and such, can be appropriately used as theantigen-binding domain of the antigen-binding molecule. Such geneticallyrecombinant antibodies include, for example, humanized antibodies. Thesealtered antibodies are appropriately produced by known methods.

An antibody variable region used to produce the antigen-binding domainof an antigen-binding molecule described herein is generally formed bythree complementarity-determining regions (CDRs) that are separated byfour framework regions (FRs). CDR is a region that substantiallydetermines the binding specificity of an antibody. The amino acidsequences of CDRs are highly diverse. On the other hand, the FR-formingamino acid sequences often have high identity even among antibodies withdifferent binding specificities. Therefore, generally, the bindingspecificity of a certain antibody can be introduced to another antibodyby CDR grafting.

A humanized antibody is also called a reshaped human antibody.Specifically, humanized antibodies prepared by grafting the CDR of anon-human animal antibody such as a mouse antibody to a human antibodyand such are known. Common genetic engineering techniques for obtaininghumanized antibodies are also known. Specifically, for example, overlapextension PCR is known as a method for grafting a mouse antibody CDR toa human FR. In overlap extension PCR, a nucleotide sequence encoding amouse antibody CDR to be grafted is added to primers for synthesizing ahuman antibody FR. Primers are prepared for each of the four FRs. It isgenerally considered that when grafting a mouse CDR to a human FR,selecting a human FR that has high identity to a mouse FR isadvantageous for maintaining the CDR function. That is, it is generallypreferable to use a human FR comprising an amino acid sequence which hashigh identity to the amino acid sequence of the FR adjacent to the mouseCDR to be grafted.

Nucleotide sequences to be ligated are designed so that they will beconnected to each other in frame. Human FRs are individually synthesizedusing the respective primers. As a result, products in which the mouseCDR-encoding DNA is attached to the individual FR-encoding DNAs areobtained. Nucleotide sequences encoding the mouse CDR of each productare designed so that they overlap with each other. Then, complementarystrand synthesis reaction is conducted to anneal the overlapping CDRregions of the products synthesized using a human antibody gene astemplate. Human FRs are ligated via the mouse CDR sequences by thisreaction.

The full length V region gene, in which three CDRs and four FRs areultimately ligated, is amplified using primers that anneal to its 5′- or3′-end, which are added with suitable restriction enzyme recognitionsequences. An expression vector for humanized antibody can be producedby inserting the DNA obtained as described above and a DNA that encodesa human antibody C region into an expression vector so that they willligate in frame. After the recombinant vector is transfected into a hostto establish recombinant cells, the recombinant cells are cultured, andthe DNA encoding the humanized antibody is expressed to produce thehumanized antibody in the cell culture (see, European Patent PublicationNo. EP 239400 and International Patent Publication No. WO 1996/002576).

By qualitatively or quantitatively measuring and evaluating theantigen-binding activity of the humanized antibody produced as describedabove, one can suitably select human antibody FRs that allow CDRs toform a favorable antigen-binding site when ligated through the CDRs.Amino acid residues in FRs may be substituted as necessary, so that theCDRs of a reshaped human antibody form an appropriate antigen-bindingsite. For example, amino acid sequence mutations can be introduced intoFRs by applying the PCR method used for grafting a mouse CDR into ahuman FR. More specifically, partial nucleotide sequence mutations canbe introduced into primers that anneal to the FR. Nucleotide sequencemutations are introduced into the FRs synthesized by using such primers.Mutant FR sequences having the desired characteristics can be selectedby measuring and evaluating the activity of the amino acid-substitutedmutant antibody to bind to the antigen by the above-mentioned method(Cancer Res. (1993) 53: 851-856).

Alternatively, desired human antibodies can be obtained by immunizingtransgenic animals having the entire repertoire of human antibody genes(see WO 1993/012227; WO 1992/003918; WO 1994/002602; WO 1994/025585; WO1996/034096; WO 1996/033735) by DNA immunization.

Furthermore, techniques for preparing human antibodies by panning usinghuman antibody libraries are also known. For example, the V region of ahuman antibody is expressed as a single-chain antibody (scFv) on phagesurface by the phage display method. Phages expressing an scFv thatbinds to the antigen can be selected. The DNA sequence encoding thehuman antibody V region that binds to the antigen can be determined byanalyzing the genes of selected phages. The DNA sequence of the scFvthat binds to the antigen is determined. An expression vector isprepared by fusing the V region sequence in frame with the C regionsequence of a desired human antibody, and inserting this into anappropriate expression vector. The expression vector is introduced intocells appropriate for expression such as those described above. Thehuman antibody can be produced by expressing the human antibody-encodinggene in the cells. These methods are already known (see WO 1992/001047;WO 1992/020791; WO 1993/006213; WO 1993/011236; WO 1993/019172; WO1995/001438; WO 1995/015388).

In addition to the techniques described above, techniques of B cellcloning (identification of each antibody-encoding sequence, cloning andits isolation; use in constructing expression vector in order to prepareeach antibody (IgG1, IgG2, IgG3, or IgG4 in particular); and such) suchas described in Bernasconi et al. (Science (2002) 298: 2199-2202) or inWO 2008/081008 can be appropriately used to isolate antibody genes.

EU Numbering and Kabat Numbering

According to the methods used in the present invention, amino acidpositions assigned to antibody CDR and FR are specified according toKabat's numbering (Sequences of Proteins of Immunological Interest(National Institute of Health, Bethesda, Md., 1987 and 1991)). Herein,when an antigen-binding molecule is an antibody or antigen-bindingfragment, variable region amino acids are indicated by Kabat numbering,while constant region amino acids are indicated by EU numbering based onKabat's amino acid positions.

Antigen-Binding Domains Dependent on a Target Tissue-Specific Compound

To obtain an antigen-binding domain (or an antigen-binding moleculecontaining the domain) whose antigen-binding activity varies dependingon the concentration of a target tissue-specific compound, or morespecifically, an antigen-binding domain (or an antigen-binding moleculecontaining the domain) dependent on a target tissue-specific compound,the methods indicated in the above section on binding activity may beappropriately applied. As a non-limiting embodiment, some specificexamples of the methods are presented below. For example, to confirmthat the antigen-binding activity of an antigen-binding domain (or anantigen-binding molecule containing the domain) in the presence of atarget tissue-specific compound becomes higher than the antigen-bindingactivity of an antigen-binding domain (or an antigen-binding moleculecontaining the domain) in the absence of the compound, theantigen-binding activities of the antigen-binding domain (or theantigen-binding molecule containing the domain) in the presence andabsence of the target tissue-specific compound or in the presence ofhigh and low concentrations of the compound are compared. In anothernon-limiting embodiment, for example, to confirm that theantigen-binding activity of an antigen-binding domain (or anantigen-binding molecule containing the domain) in the presence of ahigh concentration of a target tissue-specific compound becomes higherthan the antigen-binding activity of an antigen-binding domain (or anantigen-binding molecule containing the domain) in the presence of a lowconcentration of the compound, the antigen-binding activities of theantigen-binding domain (or the antigen-binding molecule containing thedomain) in the presence of high and low concentrations of the targettissue-specific compound are compared.

Furthermore, in the present invention, the phrase “the antigen-bindingactivity in the presence of a target tissue-specific compound is higherthan the antigen-binding activity in the absence of the compound” can bealternatively expressed as “the antigen-binding activity of anantigen-binding domain (or an antigen-binding molecule containing thedomain) in the absence of a target tissue-specific compound is lowerthan the antigen-binding activity in the presence of the compound”.Furthermore, in the present invention, “the antigen-binding activity ofan antigen-binding domain (or an antigen-binding molecule containing thedomain) in the absence of a target tissue-specific compound is lowerthan the antigen-binding activity in the presence of the compound” maybe alternatively described as “the antigen-binding activity of anantigen-binding domain (or an antigen-binding molecule containing thedomain) in the absence of a target tissue-specific compound is weakerthan the antigen-binding activity in the presence of the compound”.

Furthermore, in the present invention, the phrase “the antigen-bindingactivity in the presence of a high concentration of a targettissue-specific compound is higher than the antigen-binding activity inthe presence of a low concentration of the compound” can bealternatively expressed as “the antigen-binding activity of anantigen-binding domain (or an antigen-binding molecule containing thedomain) in the presence of a low concentration of a targettissue-specific compound is lower than the antigen-binding activity inthe presence of a high concentration of the compound”. In the presentinvention, “the antigen-binding activity of an antigen-binding domain(or an antigen-binding molecule containing the domain) in the presenceof a low concentration of a target tissue-specific compound is lowerthan the antigen-binding activity in the presence of a highconcentration of the compound” may be alternatively described as “theantigen-binding activity of an antigen-binding domain (or anantigen-binding molecule containing the domain) in the presence of a lowconcentration of a target tissue-specific compound is weaker than theantigen-binding activity in the presence of a high concentration of thecompound”.

Conditions when measuring antigen-binding activity other than theconcentration of a target tissue-specific compound are not particularlylimited, and can be selected appropriately by those skilled in the art.For example, it is possible to measure under conditions of HEPES bufferand 37° C. For example, Biacore (GE Healthcare) or such can be used formeasurement. When the antigen is a soluble molecule, the activity of anantigen-binding domain (or an antigen-binding molecule containing thedomain) to bind to the soluble molecule can be determined by loading theantigen as an analyte onto a chip immobilized with the antigen-bindingdomain (or an antigen-binding molecule containing the domain).Alternatively, when the antigen is a membrane-type molecule, the bindingactivity towards the membrane-type molecule can be determined by loadingthe antigen-binding domain (or an antigen-binding molecule containingthe domain) as an analyte onto a chip immobilized with the antigen.

As long as the antigen-binding activity of an antigen-binding domain (oran antigen-binding molecule containing the domain) contained inantigen-binding molecules of the present invention in the absence of atarget tissue-specific compound is weaker than the antigen-bindingactivity in the presence of the target tissue-specific compound, theratio between the antigen-binding activity in the absence of thecompound and the antigen-binding activity in the presence of thecompound is not particularly limited. However, the value of KD (in theabsence of the compound)/KD (in the presence of the compound), which isa ratio of dissociation constant (KD) against an antigen in the absenceof the target tissue-specific compound to KD in the presence of thecompound, is preferably 2 or greater, more preferably 10 or greater, andstill more preferably 40 or greater. The upper limit of the value of KD(in the absence of the compound)/KD (in the presence of the compound) isnot particularly limited, and may be any value, for example, 400, 1,000,or 10,000, as long as it can be provided by the technologies of thoseskilled in the art. When antigen-binding activity is not observed in theabsence of the target tissue-specific compound, the value of the upperlimit is infinity.

As long as the antigen-binding activity of an antigen-binding domain (oran antigen-binding molecule containing the domain) contained inantigen-binding molecules of the present invention in the presence of alow concentration of a target tissue-specific is weaker than theantigen-binding activity in the presence of a high concentration of thetarget tissue-specific compound, the ratio between the antigen-bindingactivity in the presence of a low concentration of the compound and theantigen-binding activity in the presence of a high concentration of thecompound is not particularly limited. However, the value of KD (in thepresence of a low concentration of the compound)/KD (in the presence ofa high concentration of the compound), which is a ratio of dissociationconstant (KD) against an antigen in the presence of a low concentrationof the target tissue-specific compound to KD in the presence of a highconcentration of the compound, is preferably 2 or greater, morepreferably 10 or greater, and still more preferably 40 or greater. Theupper limit of the value of KD (in the presence of a low concentrationof the compound)/KD (in the presence of a high concentration of thecompound) is not particularly limited, and may be any value, forexample, 400, 1,000, or 10,000, as long as it can be provided by thetechnologies of those skilled in the art. When antigen-binding activityis not observed in the presence of a low concentration of the targettissue-specific compound, the value of the upper limit is infinity.

For the value of antigen-binding activity, if the antigen is a solublemolecule, dissociation constant (KD) can be used; and if the antigen isa membrane-type molecule, apparent dissociation constant (apparent KD)can be used. The dissociation constant (KD) and apparent dissociationconstant (apparent KD) can be determined by methods known to thoseskilled in the art, for example, using Biacore (GE Healthcare), aScatchard plot, a flow cytometer, or such.

As another indicator that shows the ratio between the antigen-bindingactivity of an antigen-binding domain (or an antigen-binding moleculecontaining the domain) of the present invention in the absence of atarget tissue-specific compound and the antigen-binding activity in thepresence of the compound, for example, dissociation rate constant kd canbe suitably used. When the dissociation rate constant (kd) is usedinstead of the dissociation constant (KD) as an indicator that shows thebinding activity ratio, the value of kd (in the absence of thecompound)/kd (in the presence of the compound), which is a ratio betweenkd (dissociation rate constant) for an antigen in the absence of atarget tissue-specific compound and kd in the presence of the compound,is preferably 2 or greater, more preferably 5 or greater, even morepreferably 10 or greater, and still more preferably 30 or greater. Theupper limit of the value of kd (in the absence of the compound)/kd (inthe presence of the compound) is not particularly limited, and may beany value, for example, 50, 100, or 200, as long as it can be providedby the common technical knowledge of those skilled in the art. Whenantigen-binding activity is not observed in the absence of thetissue-specific compound, there is no dissociation and the value of theupper limit becomes infinity.

As another indicator that shows the ratio between the antigen-bindingactivity of an antigen-binding domain (or an antigen-binding moleculecontaining the domain) of the present invention in the presence of a lowconcentration of a target tissue-specific compound and theantigen-binding activity in the presence of a high concentration of thecompound, for example, dissociation rate constant kd can be suitablyused. When the dissociation rate constant (kd) is used instead of thedissociation constant (KD) as an indicator showing the binding activityratio, the value of kd (in the presence of a low concentration of thecompound)/kd (in the presence of a high concentration of the compound),which is a ratio between kd (dissociation rate constant) for an antigenin the presence of a low concentration of a target tissue-specificcompound and kd in the presence of a high concentration of the compound,is preferably 2 or greater, more preferably 5 or greater, even morepreferably 10 or greater, and still more preferably 30 or greater. Theupper limit of the value of kd (in the presence of a low concentrationof the compound)/kd (in the presence of a high concentration of thecompound) is not particularly limited, and may be any value, forexample, 50, 100, or 200, as long as it can be provided by the commontechnical knowledge of those skilled in the art. When antigen-bindingactivity is not observed in the presence of a low concentration of thetarget tissue-specific compound, there is no dissociation and the valueof the upper limit becomes infinity.

For the value of antigen-binding activity, if the antigen is a solublemolecule, dissociation rate constant (kd) can be used; and if theantigen is a membrane-type molecule, apparent dissociation rate constant(apparent kd) can be used. The dissociation rate constant (kd) andapparent dissociation rate constant (apparent kd) can be determined bymethods known to those skilled in the art, for example, using Biacore(GE Healthcare), a flow cytometer, or such. In the present invention,when measuring the antigen-binding activity of an antigen-binding domain(or an antigen-binding molecule containing the domain) at a certainconcentration of the target tissue-specific compound, conditions otherthan the concentration of the compound concentration are preferably thesame.

For example, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by screening of antigen-binding domains (or antigen-bindingmolecules) that comprises the steps of:

(a) determining antigen-binding activity of antigen-binding domains (orantigen-binding molecules) in the absence of a target tissue-specificcompound;

(b) determining antigen-binding activity of the antigen-binding domains(or antigen-binding molecules) in the presence of the targettissue-specific compound; and

(c) selecting an antigen-binding domain (or an antigen-binding molecule)with lower antigen-binding activity in the absence of the targettissue-specific compound than in the presence of the compound.

For example, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by screening ofantigen-binding domains (or antigen-binding molecules) that comprisesthe steps of:

(a) determining antigen-binding activity of antigen-binding domains (orantigen-binding molecules) in the presence of a low concentration of atarget tissue-specific compound;

(b) determining antigen-binding activity of the antigen-binding domains(or antigen-binding molecules) in the presence of a high concentrationof the target tissue-specific compound; and

(c) selecting an antigen-binding domain (or an antigen-binding molecule)with lower antigen-binding activity in the presence of a lowconcentration of the target tissue-specific compound than in thepresence of a high concentration of the compound.

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by screening of antigen-binding domains (or antigen-bindingmolecules) or a library thereof that comprises the steps of:

(a) contacting antigen-binding domains (or antigen-binding molecules) ora library thereof with an antigen in the presence of a targettissue-specific compound;

(b) placing antigen-binding domains (or antigen-binding molecules) thatbind to the antigen in said step (a) in the absence of the compound;

(c) isolating an antigen-binding domain (or an antigen-binding molecule)that dissociated in said step (b).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by screening ofantigen-binding domains (or antigen-binding molecules) or a librarythereof that comprises the steps of:

(a) contacting antigen-binding domains (or antigen-binding molecules) ora library thereof with an antigen in the presence of a highconcentration of a target tissue-specific compound;

(b) placing antigen-binding domains (or antigen-binding molecules) thatbind to the antigen in said step (a) in the presence of a lowconcentration of the compound;

(c) isolating an antigen-binding domain (or an antigen-binding molecule)that dissociates in said step (b).

Alternatively, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by screening of antigen-binding domains (or antigen-bindingmolecules) or a library thereof that comprises the steps of:

(a) contacting a library of antigen-binding domains (or antigen-bindingmolecules) with an antigen in the absence of a target tissue-specificcompound;

(b) selecting antigen-binding domains (or antigen-binding molecules)that do not bind to the antigen in said step (a);

(c) allowing the antigen-binding domains (or antigen-binding molecules)selected in said step (b) to bind to the antigen in the presence of thecompound; and

(d) isolating an antigen-binding domain (or an antigen-binding molecule)that binds to the antigen in said step (c).

Alternatively, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by screening ofantigen-binding domains (or antigen-binding molecules) or a librarythereof that comprises the steps of:

(a) contacting a library of antigen-binding domains (or antigen-bindingmolecules) with an antigen in the presence of a low concentration of atarget tissue-specific compound;

(b) selecting antigen-binding domains (or antigen-binding molecules)that do not bind to the antigen in said step (a);

(c) allowing the antigen-binding domains (or antigen-binding molecules)selected in said step

(b) to bind to the antigen in the presence of a high concentration thecompound; and

(d) isolating an antigen-binding domain (or an antigen-binding molecule)that binds to the antigen in said step (c).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by a screening method comprising the steps of:

(a) contacting a library of antigen-binding domains (or antigen-bindingmolecules) with an antigen-immobilized column in the presence of atarget tissue-specific compound;

(b) eluting an antigen-binding domain (or antigen-binding molecule) thatbinds to the column in said step (a) from the column in the absence ofthe compound; and

(c) isolating the antigen-binding domain (or antigen-binding molecule)eluted in said step (b).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by a screeningmethod comprising the steps of:

(a) contacting a library of antigen-binding domains (or antigen-bindingmolecules) with an antigen-immobilized column in the presence of a highconcentration of a target tissue-specific compound;

(b) eluting an antigen-binding domain (or antigen-binding molecule) thatbinds to the column in said step (a) from the column in the presence ofa low concentration of the compound; and

(c) isolating the antigen-binding domain (or antigen-binding molecule)eluted in said step (b).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by a screening method comprising the steps of:

(a) allowing a library of antigen-binding domains (or antigen-bindingmolecules) to pass through an antigen-immobilized column in the absenceof a target tissue-specific compound;

(b) collecting an antigen-binding domain (or antigen-binding molecule)eluted without binding to the column in said step (a);

(c) allowing the antigen-binding domain (or antigen-binding molecule)collected in said step (b) to bind to the antigen in the presence of thecompound; and

(d) isolating an antigen-binding domain (or antigen-binding molecule)that binds to the antigen in said step (c).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by a screeningmethod comprising the steps of:

(a) allowing a library of antigen-binding domains (or antigen-bindingmolecules) to pass through an antigen-immobilized column in the presenceof a low concentration of a target tissue-specific compound;

(b) collecting an antigen-binding domain (or antigen-binding molecule)eluted without binding to the column in said step (a);

(c) allowing the antigen-binding domain (or antigen-binding molecule)collected in said step (b) to bind to the antigen in the presence of ahigh concentration of the compound; and

(d) isolating an antigen-binding domain (or antigen-binding molecule)that binds to the antigen in said step (c).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound, may beobtained by a screening method comprising the steps of:

(a) contacting an antigen with a library of antigen-binding domains (orantigen-binding molecules) in the presence of a target tissue-specificcompound;(b) obtaining an antigen-binding domain (or antigen-binding molecule)that binds to the antigen in said step (a);(c) placing the antigen-binding domain (or antigen-binding molecule)obtained in said step (b) in the absence of the compound; and(d) isolating an antigen-binding domain (or antigen-binding molecule)whose antigen-binding activity in said step (c) is weaker than that ofthe reference selected in said step (b).

Furthermore, in an embodiment provided by the present invention, anantigen-binding domain (or an antigen-binding molecule containing thedomain) with lower antigen-binding activity in the presence of a lowconcentration of a target tissue-specific compound than in the presenceof a high concentration of the compound, may be obtained by a screeningmethod comprising the steps of:

(a) contacting an antigen with a library of antigen-binding domains (orantigen-binding molecules) in the presence of a high concentration of atarget tissue-specific compound;(b) obtaining an antigen-binding domain (or antigen-binding molecule)that binds to the antigen in said step (a);(c) placing the antigen-binding domain (or antigen-binding molecule)obtained in said step (b) in the presence of a low concentration of thecompound; and(d) isolating an antigen-binding domain (or antigen-binding molecule)whose antigen-binding activity in said step (c) is weaker than that ofthe reference selected in said step (b).

The above-mentioned steps may be repeated two or more times. Thus, thepresent invention provides an antigen-binding domain (or anantigen-binding molecule containing the domain) with lowerantigen-binding activity in the absence of a target tissue-specificcompound than in the presence of the compound, or an antigen-bindingdomain (or an antigen-binding molecule containing the domain) with lowerantigen-binding activity in the presence of a low concentration of atarget tissue-specific compound than in the presence of a highconcentration of the compound, obtained by screening methods thatfurther comprise the step of repeating steps (a) to (c) or (a) to (d)two or more times in the above-mentioned screening methods. The numberof repeats of steps (a) to (c) or (a) to (d) is not particularlylimited, and it is generally ten or less.

In the screening methods of the present invention, a targettissue-specific compound may be a compound defined by quantitativetarget tissue specificity such as presence in the target tissue at aconcentration (for example, high concentration or low concentration)different from the concentration in non-target tissues. For example, atarget tissue-specific compound is differentially present at anyconcentrations. However, generally, a target tissue-specific compoundcan be present at a concentration increased by at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 100%, at least 110%, at least120%, at least 130%, at least 140%, at least 150%, at least 2-fold, atleast 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, atleast 10³-fold, at least 10⁴-fold, at least 10⁵-fold, at least 10⁶-fold,or more, or up to infinity (when the compound is absent in non-targettissues).

The threshold differentiating low and high concentrations can be setappropriately according to the compound. For example, in a non-limitingembodiment of the threshold of ATP or adenosine, the threshold for alow-concentration condition may be selected appropriately from thevalues of 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, and 0 M. Depending on thepredetermined threshold, the high-concentration condition may be setappropriately at a value selected from at least 110%, at least 120%, atleast 130%, at least 140%, at least 150%, at least twice, at leastfive-fold, at least 10-fold, at least 50-fold, at least 100-fold, atleast 10³-fold, at least 10⁴-fold, at least 10⁵-fold, and at least10⁶-fold the value of each threshold. Furthermore, in a non-limitingembodiment of PGE2, the threshold for a low-concentration condition maybe selected appropriately from the values of 10 pM, 1 pM, 100 fM, 10 fM,1 fM, and 0 M. Depending on the predetermined threshold, thehigh-concentration condition may be set appropriately at a valueselected from at least 110%, at least 120%, at least 130%, at least140%, at least 150%, at least twofold, at least five-fold, at least10-fold, at least 50-fold, at least 100-fold, at least 10³-fold, atleast 10⁴-fold, at least 10⁵-fold, and at least 10⁶-fold the value ofeach threshold. Furthermore, in a non-limiting embodiment of Kynurenine,the threshold for a low-concentration condition may be selectedappropriately from the values of 10 μM, 1 μM, 100 nM, 10 nM, and 1 nM,and 0 M. Depending on the predetermined threshold, thehigh-concentration condition may be set appropriately at a valueselected from at least 110%, at least 120%, at least 130%, at least140%, at least 150%, at least twofold, at least five-fold, at least10-fold, at least 50-fold, at least 100-fold, at least 10³-fold, atleast 10⁴-fold, at least 10⁵-fold, and at least 10⁶-fold the value ofeach threshold.

The antigen-binding activity of an antigen-binding domain (or anantigen-binding molecule) may be measured by a method known to thoseskilled in the art, and conditions other than the concentration of atarget tissue-specific compound can be set appropriately by one skilledin the art. The antigen-binding activity of an antigen-binding domain(or an antigen-binding molecule) can be assessed as dissociationconstant (KD), apparent dissociation constant (apparent KD),dissociation rate constant (kd), apparent dissociation rate constant(apparent kd), etc. They can be determined by methods known to thoseskilled in the art, for example, using Biacore (GE Healthcare), theScatchard plot, FACS, or such.

In the present invention, the step of selecting an antibody or anantigen-binding domain with higher antigen-binding activity in thepresence of a target tissue-specific compound than in the absence of thecompound has the same meaning as the step of selecting an antibody or anantigen-binding domain with lower antigen-binding activity in theabsence of a target tissue-specific compound than in the presence of thecompound.

In the present invention, the step of selecting an antibody or anantigen-binding domain with higher antigen-binding activity in thepresence of a high concentration of a target tissue-specific compoundthan in the presence of a low concentration of the compound has the samemeaning as the step of selecting an antibody or an antigen-bindingdomain with lower antigen-binding activity in the absence of a targettissue-specific compound than in the presence of the compound.

As long as antigen-binding activity in the absence of a targettissue-specific compound is lower than the antigen-binding activity inthe presence of the compound, the difference between antigen-bindingactivity in the presence of the compound and antigen-binding activity inthe absence of the compound is not particularly limited, but preferably,the antigen-binding activity in the presence of the compound relative tothe antigen-binding activity in the absence of the compound is twofoldor more, more preferably 10-fold or more, and even more preferably40-fold or more. The upper limit of the difference between theantigen-binding activities is not particularly limited, and as long asit can be produced by the techniques of those skilled in the art, anyvalue such as 400-fold, 1000-fold, or 10000-fold is possible. In theabsence of a target tissue-specific compound, when antigen-bindingactivity is not observed, this upper limit becomes infinity.

The antigen-binding domains (or antigen-binding molecules containing thedomains) of the present invention which are to be screened by theaforementioned screening methods may be any antigen-binding domains (orantigen-binding molecules); and for example, the above-mentionedantigen-binding domains (or antigen-binding molecules) can be screened.For example, antigen-binding domains (or antigen-binding molecules)having naturally-occurring sequences can be screened, andantigen-binding domains (or antigen-binding molecules) with substitutedamino acid sequences may be screened.

Library

According to a certain embodiment, the antigen-binding domain (or anantigen-binding molecule containing this domain) of the presentinvention can be obtained from a library mainly comprising a pluralityof antigen-binding molecules having different sequences from oneanother, in which at least one amino acid residue that changes thebinding activity of the antigen-binding molecule toward an antigendependent on a target tissue-specific compound is contained in theantigen-binding domain. Examples of the compound include (1) primarymetabolites of the Krebs cycle or the glycolytic pathway such aslactose, succinic acid, or citric acid, (2) amino acids such as alanine,glutamic acid, or asparagine, (3) kynurenine and amino acid metabolitesthereof such as anthranilic acid, 3-hudroxykynurenine, and kynurenicacid, (4) arachidonic acid metabolites such as prostaglandin E2, and (5)nucleosides carrying a purine ring structure such as adenosine,adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosinemonophosphate (AMP). Below are examples of such a library mainlycomprising a plurality of antigen-binding molecules having differentsequences from one another, in which at least one amino acid residuethat changes the binding activity of the antigen-binding molecule towardadenosine- and/or ATP-dependent antigens which are targettissue-specific compounds is contained in the antigen-binding domain.

Herein, a “library” refers to a plurality of antigen-binding moleculesor a plurality of fusion polypeptides containing antigen-bindingmolecules, or nucleic acids or polynucleotides encoding their sequences.The sequences of a plurality of antigen-binding molecules or a pluralityof fusion polypeptides containing antigen-binding molecules in a libraryare not identical, but are different from one another.

Herein, the phrase “sequences are different from one another” in theexpression “a plurality of antigen-binding molecules whose sequences aredifferent from one another” means that the sequences of antigen-bindingmolecules in a library are different from one another. Specifically, ina library, the number of sequences different from one another reflectsthe number of independent clones with different sequences, and may alsobe referred to as “library size”. The library size of a conventionalphage display library ranges from 10⁶ to 10¹². The library size can beincreased up to 10¹⁴ by the use of known techniques such as ribosomedisplay. However, the actual number of phage particles used in panningselection of a phage library is in general 10 to 10,000 times greaterthan the library size. This excess multiplicity is also referred to as“the number of library equivalents”, and means that there are 10 to10,000 individual clones that have the same amino acid sequence. Thus,in the present invention, the phrase “sequences are different from oneanother” means that the sequences of independent antigen-bindingmolecules in a library, excluding library equivalents, are differentfrom one another. More specifically, the above means that there are 10⁶to 10¹⁴ antigen-binding molecules whose sequences are different from oneanother, preferably 10⁷ to 10¹² molecules, more preferably 10⁸ to 10¹¹molecules, and particularly preferably 10⁸ to 10¹⁰ molecules whosesequences are different from one another.

Herein, the phrase “a plurality of” in the expression “a library mainlycomposed of a plurality of antigen-binding molecules” generally refersto, in the case of, for example, antigen-binding molecules, fusionpolypeptides, polynucleotide molecules, vectors, or viruses of thepresent invention, a group of two or more types of the substance. Forexample, when two or more substances are different from one another in aparticular characteristic, this means that there are two or more typesof the substance. Such examples may include, for example, mutant aminoacids observed at specific amino acid positions in an amino acidsequence. For example, when there are two or more antigen-bindingmolecules of the present invention whose sequences are substantially thesame or preferably the same except for flexible residues or except forparticular mutant amino acids at hypervariable positions exposed on thesurface, there are a plurality of antigen-binding molecules of thepresent invention. In another example, when there are two or morepolynucleotide molecules whose sequences are substantially the same orpreferably the same except for nucleotides encoding flexible residues ornucleotides encoding mutant amino acids of hypervariable positionsexposed on the surface, there are a plurality of polynucleotidemolecules of the present invention.

In addition, herein, the phrase “mainly composed of” in the expression“a library mainly composed of a plurality of antigen-binding molecules”reflects the number of antigen-binding molecules whose antigen-bindingactivity varies depending on the concentration of a targettissue-specific compound, among independent clones with differentsequences in a library. Specifically, it is preferable that there are atleast 10⁴ antigen-binding molecules having such binding activity in alibrary. More preferably, antigen-binding domains of the presentinvention can be obtained from a library containing at least 10⁵antigen-binding molecules having such binding activity. Still morepreferably, antigen-binding domains of the present invention can beobtained from a library containing at least 10⁶ antigen-bindingmolecules having such binding activity. Particularly preferably,antigen-binding domains of the present invention can be obtained from alibrary containing at least 10⁷ antigen-binding molecules having suchbinding activity. Yet more preferably, antigen-binding domains of thepresent invention can be obtained from a library containing at least 10⁸antigen-binding molecules having such binding activity. Alternatively,this may also be preferably expressed as the ratio of the number ofantigen-binding molecules in which antigen-binding activity of theantigen-binding domain varies depending on the presence or absence ofadenosine and/or ATP with respect to the number of independent cloneshaving different sequences in a library. Specifically, antigen-bindingdomains of the present invention can be obtained from a library in whichantigen-binding molecules having such binding activity account for 0.1%to 80%, preferably 0.5% to 60%, more preferably 1% to 40%, still morepreferably 2% to 20%, and particularly preferably 4% to 10% ofindependent clones with different sequences in the library. In the caseof fusion polypeptides, polynucleotide molecules, or vectors, similarexpressions may be possible using the number of molecules or the ratioto the total number of molecules. In the case of viruses, similarexpressions may also be possible using the number of virions or theratio to total number of virions.

Amino Acids that Change the Antigen-Binding Activity of theAntigen-Binding Domain Depending on the Presence or Absence of Adenosineand/or ATP

Antigen-binding domains or antibodies of the present invention screenedby the above-described screening methods may be prepared in any manner.It is possible to use preexisting antibodies, preexisting libraries(phage libraries, etc.), antibodies or libraries prepared fromhybridomas obtained by immunizing animals or from B cells of immunizedanimals, and antibodies or libraries prepared from immune cells such asB cells of animals immunized by a conjugate in which adenosine or ATP issuitably linked to an adjuvant agent such as a highly immunogenic T cellepitope peptide. A non-limiting example of the T cell epitope peptidesuitably includes Tetanus toxin-derived p30 helper peptide (shown in SEQID NO: 4, and also referred to as Fragment C (FrC)).

Examples of amino acids that change the antigen-binding activity of theantigen-binding molecule depending on the presence or absence ofadenosine and/or ATP as described above include amino acids that form anadenosine- and/or ATP-binding motif. The amino acid positions where theabove-mentioned amino acids are contained in the antigen-binding domainare not limited to any specific position. As long as the antigen-bindingactivity of the antigen-binding domain is changed depending on thepresence or absence of adenosine and/or ATP, any position in the heavychain variable region or light chain variable region forming theantigen-binding domain is possible. More specifically, theantigen-binding domains of the present invention may be obtained from alibrary mainly comprising antigen-binding molecules having differentsequences from one another, in which the amino acids that change theantigen-binding activity of the antigen-binding molecule depending onthe presence or absence of adenosine and/or ATP are contained in theantigen-binding domain of the heavy chain. In a non-limiting embodiment,antigen-binding domains of the present invention may be obtained from alibrary mainly comprising antigen-binding molecules having differentsequences from one another, in which the amino acids that change theantigen-binding activity of the antigen-binding molecule depending onthe presence or absence of adenosine and/or ATP are contained in CDR1,CDR2, and/or CDR3 of the heavy chain. In another non-limitingembodiment, antigen-binding domains of the present invention may beobtained from a library mainly comprising antigen-binding moleculeshaving different sequences from one another, in which the amino acidsthat change the antigen-binding activity of the antigen-binding moleculedepending on the presence or absence of adenosine and/or ATP arecontained in FR1, FR2, FR3 and/or FR4 of the heavy chain.

Furthermore, in an embodiment of the present invention, antigen-bindingdomains of the present invention may be obtained from a library mainlycomprising antigen-binding molecules having different sequences from oneanother, in which the amino acids that change the antigen-bindingactivity of the antigen-binding molecule depending on the presence orabsence of adenosine and/or ATP are contained in the antigen-bindingdomain of the heavy chain and/or light chain. In a non-limitingembodiment, antigen-binding domains of the present invention may beobtained from a library mainly comprising antigen-binding moleculeshaving different sequences from one another, in which the amino acidsthat change the antigen-binding activity of the antigen-binding moleculedepending on the presence or absence of adenosine and/or ATP arecontained in CDR1, CDR2, and/or CDR3 of the heavy chain and/or lightchain. In another non-limiting embodiment, antigen-binding domains ofthe present invention may be obtained from a library mainly comprisingantigen-binding molecules having different sequences from one another,in which the amino acids that change the antigen-binding activity of theantigen-binding molecule depending on the presence or absence ofadenosine and/or ATP are contained in FR1, FR2, FR3 and/or FR4 of theheavy chain and/or light chain.

In a non-limiting embodiment, examples of such amino acids include anyone or more amino acids selected from amino acids at positions 52, 52a,53, 96, 100a, and 100c contained in the heavy chain variable region.Also, in a non-limiting embodiment, examples of such amino acids includeone or more amino acids selected from amino acids including Ser atposition 52, Ser at position 52a, Arg at position 53, Gly at position96, Leu at position 100a, and Trp at position 100c contained in theheavy chain variable region.

Any framework sequence can be used as the framework sequence of thelight-chain and/or heavy-chain variable regions of an antigen-bindingmolecule as long as the amino acids that change the antigen-bindingactivity of the antigen-binding molecule depending on the presence orabsence of adenosine and/or ATP are contained in the antigen-bindingdomain of the heavy chain and/or light chain. The origin of theframework sequences is not limited, and they may be obtained from humanor any nonhuman organisms. Such organisms preferably include mice, rats,guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep,bovines, horses, camels and organisms selected from nonhuman primates.In a particularly preferred embodiment, the framework sequences of thelight chain and/or heavy chain variable region of an antigen-bindingmolecule preferably have human germ-line framework sequences. Thus, inan embodiment of the present invention, if the entire frameworksequences are human sequences, it is thought that an antigen-bindingmolecule of the present invention induces little or no immunogenicresponse when it is administered to humans (for example, to treatdiseases). In the above sense, the phrase “containing a germ linesequence” in the present invention means that a part of the frameworksequences of the present invention is identical to a part of any humangerm line framework sequences. For example, when the heavy chain FR2sequence of an antigen-binding molecule of the present invention is acombination of heavy chain FR2 sequences of different human germ lineframework sequences, such a molecule is also an antigen-binding molecule“containing a germ line sequence” in the present invention. Even whenthe framework sequences of antigen-binding molecules of the presentinvention are sequences with substitutions, they are antigen-bindingmolecules “containing a germ line sequence” of the present invention.Examples of such sequences with substitutions include, in particular,sequences in which amino acids of part of human germ line frameworksequences have been substituted with amino acids that change theantigen-binding activity of the antigen-binding molecule depending onthe presence or absence of adenosine and/or ATP.

Preferred examples of the frameworks include, for example, fully humanframework region sequences currently known, which are included in thewebsite of V-Base (http://vbase.mrc-cpe.cam.ac.uk/) or others. Thoseframework region sequences can be appropriately used as a germ linesequence contained in an antigen-binding molecule of the presentinvention. The germ line sequences may be categorized according to theirsimilarity (Tomlinson et al. (J. Mol. Biol. (1992) 227, 776-798);Williams and Winter (Eur. J. Immunol. (1993) 23, 1456-1461); Cox et al.(Nat. Genetics (1994) 7, 162-168)). Appropriate germ line sequences canbe selected from Vκ, which is grouped into seven subgroups; Vλ, which isgrouped into ten subgroups; and VH, which is grouped into sevensubgroups.

Fully human VH sequences preferably include, but are not limited to, forexample, VH sequences of:

subgroup VH1 (for example, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24, VH1-45,VH1-46, VH1-58, and VH1-69);subgroup VH2 (for example, VH2-5, VH2-26, and VH2-70);subgroup VH3 (VH3-7, VH3-9, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20,VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49,VH3-53, VH3-64, VH3-66, VH3-72, VH3-73, and VH3-74);subgroup VH4 (VH4-4, VH4-28, VH4-31, VH4-34, VH4-39, VH4-59, andVH4-61);subgroup VH5 (VH5-51);subgroup VH6 (VH6-1); andsubgroup VH7 (VH7-4 and VH7-81).These are also described in known documents (Matsuda et al. (J. Exp.Med. (1998) 188, 1973-1975)) and such, and thus persons skilled in theart can appropriately design antigen-binding molecules of the presentinvention based on the information of these sequences. It is alsopreferable to use other fully human frameworks or framework sub-regions.

Fully human Vκ sequences preferably include, but are not limited to, forexample:

A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22, L23,L24, O2, O4, O8, O12, O14, and O18 grouped into subgroup Vk1;A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, and O11, grouped intosubgroup Vk2; A11, A27, L2, L6, L10, L16, L20, and L25, grouped intosubgroup Vk3;B3, grouped into subgroup Vk4;B2 (herein also referred to as Vk5-2), grouped into subgroup Vk5; andA10, A14, and A26, grouped into subgroup Vk6(Kawasaki et al. (Eur. J. Immunol. (2001) 31, 1017-1028); Schable andZachau (Biol. Chem. Hoppe Seyler (1993) 374, 1001-1022);Brensing-Kuppers et al. (Gene (1997) 191, 173-181)).

Fully human Vλ sequences preferably include, but are not limited to, forexample:

V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17, V1-18,V1-19, V1-20, and V1-22, grouped into subgroup VL1;V2-1, V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19,grouped into subgroup VL1;V3-2, V3-3, and V3-4, grouped into subgroup VL3;V4-1, V4-2, V4-3, V4-4, and V4-6, grouped into subgroup VL4; andV5-1, V5-2, V5-4, and V5-6, grouped into subgroup VL5 (Kawasaki et al.(Genome Res. (1997) 7, 250-261)).

Normally, these framework sequences are different from one another atone or more amino acid residues. These framework sequences can be usedin combination with “at least one amino acid residue that alters theantigen-binding activity of an antigen-binding domain depending on thepresence or absence of adenosine and/or ATP” of the present invention.Other examples of the fully human frameworks used in combination with“at least one amino acid residue that alters the antigen-bindingactivity of an antigen-binding domain depending on the presence orabsence of adenosine and/or ATP” of the present invention include, butare not limited to, for example, KOL, NEWM, REI, EU, TUR, TEI, LAY, andPOM (for example, Kabat et al. (1991) supra; Wu et al. (J. Exp. Med.(1970) 132, 211-250)).

Without being bound by a particular theory, one reason for theexpectation that the use of germ line sequences precludes adverse immuneresponses in most individuals is believed to be as follows. As a resultof the process of affinity maturation during normal immune responses,somatic mutation occurs frequently in the variable regions ofimmunoglobulin. Such mutations mostly occur around CDRs whose sequencesare hypervariable, but also affect residues of framework regions. Suchframework mutations do not exist on the germ line genes, and also theyare less likely to be immunogenic in patients. On the other hand, thenormal human population is exposed to most of the framework sequencesexpressed from the germ line genes. As a result of immunotolerance,these germ line frameworks are expected to have low or no immunogenicityin patients. To maximize the possibility of immunotolerance, variableregion-encoding genes may be selected from a group of commonly occurringfunctional germ line genes.

Known methods such as site-directed mutagenesis (Kunkel et al. (Proc.Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR canbe appropriately employed to produce the antigen-binding molecules ofthe present invention in which the above-described variable regionsequences, heavy or light chain variable region sequences, CDRsequences, or framework sequences contain amino acids that alter theantigen-binding activity of the antigen-binding domain depending on thepresence or absence of adenosine and/or ATP.

For example, a library which contains a plurality of antigen-bindingmolecules of the present invention whose sequences are different fromone another can be constructed by combining heavy chain variable regionsprepared as a randomized variable region sequence library with a lightchain variable region selected as a CDR sequence and/or frameworksequence originally containing at least one amino acid residue thatalters the antigen-binding activity of the antigen-binding domaindepending on the presence or absence of adenosine and/or ATP.

Alternatively, a heavy chain and/or light chain variable region sequenceselected as a CDR sequence and/or a framework sequence originallycontaining at least one amino acid residue that changes theantigen-binding activity of an antigen-binding domain depending on thepresence or absence of adenosine and/or ATP as mentioned above, can bedesigned to contain various amino acid residues other than the aboveamino acid residue(s). Herein, such residues are referred to as“flexible residues”. The number and position of flexible residues arenot particularly limited as long as the antigen-binding activity of theantigen-binding molecule of the present invention varies depending onthe concentration of a tissue-specific compound. Specifically, the CDRsequences and/or FR sequences of the heavy chain and/or light chain maycontain one or more flexible residues. One can identify the flexibleresidues and those residues that can be substituted into other aminoacids for library production by introducing mutations or by crystalstructure analysis of complexes formed between an antibody and adenosineand/or ATP. For example, from crystal structure analysis of complexesformed between an antibody and adenosine and/or ATP, one can identifyresidues in the antibody that are not involved in binding to adenosineand/or ATP. One can select amino acids that can maintain binding to thecompounds at an appropriate level even when the residues that have beenidentified as not being involved in binding to adenosine and/or ATP aresubstituted into other amino acids. Accordingly, it is possible todesign a library that has the selected amino acids for the selectedresidues. In this case, one can design a library mainly comprisingmultiple antigen-binding molecules to be an assembly of antigen-bindingmolecules in which residues identified as not being involved in bindingto adenosine and/or ATP have been substituted with amino acids that aredifferent from one another. That is, the combination of individualflexible residues substituted with amino acids that are different fromone another can provide sequence diversity in antigen-binding moleculescontaining the flexible residues.

Antigen-binding molecules can be designed to include residues wherein atleast one of the residues identified to be involved in binding toadenosine and/or ATP binding becomes any residue selected from theresidue and other residues that are different from the residue. In anon-limiting embodiment, examples of amino acids identified as beinginvolved in binding to adenosine and/or ATP may include one or moreamino acids selected from amino acids at positions 52, 52a, 53, 96,100a, and 100c in the heavy chain variable region. In a non-limitingembodiment, examples of such amino acids include one or more amino acidsselected from amino acids including Ser at position 52, Ser at position52a, Arg at position 53, Gly at position 96, Leu at position 100a, andTrp at position 100c contained in the heavy chain variable region. Forexample, when Leu at position 100a mentioned above is identified to beinvolved in binding to adenosine and/or ATP, the amino acid residue atposition 100a in the antigen-binding molecules included in the librarymay be any amino acid residue selected from the flexible residues ofHis, Met, Leu, Arg, Trp, or Tyr, in addition to Leu.

In a non-limiting embodiment, examples of the flexible residues mayinclude amino acids at positions 31, 32, 33, 35, 50, 55, 56, 57, 58, 59,95, 96, 97, 98, 99, 100, 100a, and 100b contained in the heavy chainvariable region. In another non-limiting embodiment, examples of suchamino acids may include amino acids at positions 26, 27, 27a, 27b, 27c,28, 29, 31, 32, 50, 51, 52, 53, 54, 55, 89, 90, 91, 92, 93, 94, 95a, 96,and 97 contained in the light chain variable region.

In a non-limiting embodiment, examples of the aforementioned flexibleresidues may include the following amino acids contained in the heavychain variable region:

Asp, Gly, Asn, Ser, Arg, or Thr for the amino acid at position 31;Ala, Phe, His, Asn, Ser, or Tyr for the amino acid at position 32;Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Ser,Arg, Trp, Val, Tyr, or Thr for the amino acid at position 33;His, Ser, Thr, Tyr, or Asn for the amino acid at position 35;Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Arg,Thr, Trp, Val, Tyr, or Ser for the amino acid at position 50;Ala, Glu, Asp, Gly, Leu, Thr, Ser, Arg, or Asn for the amino acid atposition 55;Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Thr,Trp, Val, or Tyr for the amino acid at position 56;Ala, Lys, Arg, Thr, or Ile for the amino acid at position 57;Asp, Gly, Phe, His, Ser, Thr, Tyr, or Asn for the amino acid at position58;Leu, or Tyr for the amino acid at position 59;Ala, Ile, Lys, Met, Leu, Arg, Trp, Val, Tyr, or Phe for the amino acidat position 95;Ala, Asp, Asn, or Ser for the amino acid at position 96;Ala, Asp, Gly, Ile, His, Lys, Met, Leu, Asn, Ser, Val, Tyr, or Arg forthe amino acid at position 97;Ala, Glu, Asp, Gly, Phe, Ile, His, Met, Leu, Asn, Gln, Pro, Ser, Arg,Thr, Trp, Val, Tyr, or Lys for the amino acid at position 98;Ala, Glu, Asp, Phe, His, Lys, Asn, Gln, Ser, Arg, Trp, Val, Tyr, or Glyfor the amino acid at position 99;Ala, Glu, Gly, Phe, Ile, His, Lys, Met, Leu, Asn, Gln, Pro, Ser, Arg,Thr, Trp, Val, Tyr, or Asp for the amino acid at position 100;Ala, Phe, Ile, His, Lys, Met, Arg, Trp, Val, or Tyr for the amino acidat position 100a; orAla, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Arg,Thr, Trp, Val, Tyr, or Asn for the amino acid at position 100b.

In a non-limiting embodiment, examples of the aforementioned flexibleresidues may include the following amino acids contained in the lightchain variable region:

Ala, Ser, or Thr for the amino acid at position 26;Thr or Ser for the amino acid at position 27;Gly, Asn, Thr, or Ser for the amino acid at position 27a;Asn or Asp for the amino acid at position 27b;Ile or Val for the amino acid at position 27c;Asp or Gly for the amino acid at position 28;Ala, Asp, Phe, Ser, Arg, Thr, Tyr, or Gly for the amino acid at position29;Glu, Asp, Lys, or Asn for the amino acid at position 31;Ala, Asp, Ser, Thr, or Tyr for the amino acid at position 32;Asp, Gly, Lys, Asn, Gln, Ser, Arg, Tyr, or Glu for the amino acid atposition 50;Asp, Gly, Lys, Asn, Thr, or Val for the amino acid at position 51;Ala, Asp, Asn, Thr, or Ser for the amino acid at position 52;Glu, Asp, His, Asn, Gln, Ser, Tyr, or Lys for the amino acid at position53;Lys or Arg for the amino acid at position 54;Leu or Pro for the amino acid at position 55;Ala, Gly, Phe, Leu, Asn, Gln, Thr, Val, Tyr, or Ser for the amino acidat position 89;Ala, Leu, Thr, Val, or Ser for the amino acid at position 90;Ala, Asp, Phe, His, Lys, Asn, Ser, Arg, Thr, Trp, Val, or Tyr for theamino acid at position 91;Glu, Asp, Ser, Arg, Thr, Val, Tyr, or Ala for the amino acid at position92;Ala, Asp, Ile, Asn, Ser, Arg, Thr, Val, Tyr, or Gly for the amino acidat position 93;Ala, Asp, Gly, Ile, Asn, Arg, Thr, or Ser for the amino acid at position94;Ala, Glu, Asp, Gly, Phe, Ile, His, Lys, Met, Leu, Gln, Pro, Ser, Arg,Thr, Trp, Val, Tyr, or Asn for the amino acid at position 95;Ala, Glu, Asp, Gly, Ile, His, Lys, Leu, Gln, Pro, Ser, Arg, Thr, Tyr, orAsn for the amino acid at position 95a;Ala, Asp, Gly, Phe, His, Lys, Leu, Asn, Gln, Pro, Ser, Thr, Trp, Tyr, orVal for the amino acid at position 96; orAla, Gly, Ile, Met, Leu, Ser, or Val for the amino acid at position 97.

Herein, “flexible residue” refers to amino acid residue variationspresent at hypervariable amino acid positions of light-chain andheavy-chain variable regions at which several different amino acidsexist, when the amino acid sequences of known and/or native antibodiesor antigen-binding domains are compared. The hypervariable positions aregenerally located in the CDR regions. In an embodiment, the dataprovided by Kabat, Sequences of Proteins of Immunological Interest(National Institute of Health Bethesda Md., 1987 and 1991) is useful fordetermining the hypervariable positions in known and/or nativeantibodies. Furthermore, databases on the Internet(http://vbase.mrc-cpe.cam.ac.uk/, andhttp://www.bioinf.org.uk/abs/index.html) provide many collectedsequences of human light chains and heavy chains, and their locations.The information on the sequences and locations is useful for determiningthe hypervariable positions in the present invention. According to thepresent invention, when a certain amino acid position has preferablyabout 2 to about 20, preferably about 3 to about 19, preferably about 4to about 18, preferably 5 to 17, preferably 6 to 16, preferably 7 to 15,preferably 8 to 14, preferably 9 to 13, and preferably 10 to 12 possibleamino acid residue variations, the position can be said to behypervariable. In some embodiments, a certain amino acid position mayhave preferably at least about 2, preferably at least about 4,preferably at least about 6, preferably at least about 8, preferablyabout 10, and preferably about 12 possible amino acid residuevariations.

A library of the present invention that contains a plurality ofantigen-binding molecules having different sequences from one anothercan be constructed by combining heavy chain variable regions produced asa randomized variable region sequence library with the aforementionedlight chain variable regions introduced with at least one amino acidresidue that changes the antigen-binding activity of the antigen-bindingdomains depending on the presence or absence of adenosine and/or ATP.Similarly, a library of the present invention that contains a pluralityof antigen-binding molecules having different sequences from one anothercan also be produced by combining the heavy-chain variable regionsintroduced with at least one amino acid residue that changes theantigen-binding activity of the antigen-binding domains depending on thepresence or absence of adenosine and/or ATP, and having the other aminoacid residues designed as flexible residues.

When heavy chain variable regions produced as a randomized variableregion sequence library and light chain variable regions into which atleast one amino acid residue that alters the antigen-binding activity ofan antigen-binding molecule depending on the concentration of the targettissue-specific compound has been introduced are combined as describedabove, the sequences of the light chain variable regions can be designedto contain flexible residues in the same manner as described above. Thenumber and position of such flexible residues are not particularlylimited to particular embodiments as long as the antigen-bindingactivity of antigen-binding molecules of the present invention variesdepending on the presence or absence of adenosine and/or ATP.Specifically, the CDR sequences and/or FR sequences of heavy chainand/or light chain can contain one or more flexible residues.

The preferred heavy chain variable regions to be combined include, forexample, randomized variable region libraries. Known methods arecombined as appropriate to produce a randomized variable region library.In a non-limiting embodiment of the present invention, an immune libraryconstructed based on antibody genes derived from lymphocytes of animalsimmunized with a specific antigen, patients with infections, personswith an elevated antibody titer in blood as a result of vaccination,cancer patients, or auto immune disease patients, may be preferably usedas a randomized variable region library.

In another non-limiting embodiment of the present invention, a syntheticlibrary produced by replacing the CDR sequences of V genes in genomicDNA or functional reshaped V genes with a set of syntheticoligonucleotides containing sequences encoding codon sets of anappropriate length can also be preferably used as a randomized variableregion library. In this case, since sequence diversity is observed inthe heavy chain CDR3 sequence, it is also possible to replace the CDR3sequence only. A criterion of giving rise to diversity in amino acids inthe variable region of an antigen-binding molecule is that diversity isgiven to amino acid residues at surface-exposed positions in theantigen-binding molecule. The surface-exposed position refers to aposition that is considered to be able to be exposed on the surfaceand/or contacted with an antigen, based on structure, ensemble ofstructures, and/or modeled structure of an antigen-binding molecule. Ingeneral, such positions are CDRs. Preferably, surface-exposed positionsare determined using coordinates from a three-dimensional model of anantigen-binding molecule using a computer program such as the InsightIIprogram (Accelrys). Surface-exposed positions can be determined usingalgorithms known in the art (for example, Lee and Richards (J. Mol.Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst. (1983) 16,548-558)). Determination of surface-exposed positions can be performedusing software suitable for protein modeling and three-dimensionalstructural information obtained from an antibody. Software that can beused for these purposes preferably includes SYBYL Biopolymer Modulesoftware (Tripos Associates). Generally or preferably, when an algorithmrequires a user input size parameter, the “size” of a probe which isused in the calculation is set at about 1.4 Angstrom or smaller inradius. Furthermore, methods for determining surface-exposed regions andareas using software for personal computers are described by Pacios(Comput. Chem. (1994) 18 (4), 377-386; J. Mol. Model. (1995) 1, 46-53).

Furthermore, in a non-limiting embodiment of the present invention,amino acids of the variable region including the CDR region and/or theframework region may be altered appropriately to improve antibodystability. In a non-limiting embodiment, examples of such amino acidsmay include the amino acids of positions 1, 5, 10, 30, 48, and 58. Morespecifically, examples may include Gln at position 1, Gln at position 5,Asp at position 10, Asn at position 30, Leu at position 48, and Asn atposition 58. For the improvement of antibody stability, these aminoacids can be substituted for corresponding amino acids contained in agerm-line sequence. In a non-limiting embodiment, an example of such agerm line sequence may be the VH3-21 sequence. In this case, Gln ofposition 1 may be substituted with Glu, Gln of position 5 may besubstituted with Val, Asp of position 10 may be substituted with Gly,Asn of position 30 may be substituted with Ser, Leu of position 48 maybe substituted with Val, and Asn of position 58 may be substituted withTyr.

In another non-limiting embodiment of the present invention, a naivelibrary which is constructed from antibody genes derived fromlymphocytes of healthy individuals and consists of naive sequences whichare antibody sequences that do not have bias in their repertoire, canalso be particularly preferably used as a randomized variable regionlibrary (Gejima et al. (Human Antibodies (2002) 11, 121-129); Cardoso etal. (Scand. J. Immunol. (2000) 51, 337-344)). Herein, “an amino acidsequence comprising a naive sequence” refers to an amino acid sequenceobtained from such a naive library.

Fc Region

An Fc region contains an amino acid sequence derived from the heavychain constant region of an antibody. An Fc region is a portion of theantibody heavy chain constant region that includes the N terminal end ofthe hinge region, which is the papain cleavage site, at an amino acidaround position 216 (indicated by EU numbering), and the hinge, CH2, andCH3 domains. Fc regions can be obtained from human IgG1; however, theyare not limited to any specific IgG subclass. Preferred examples of theFc regions include Fc regions having FcRn-binding activity in an acidicpH range as described below. Preferred examples of the Fc regionsinclude Fc regions having Fcγ receptor-binding activity as describedbelow. In a non-limiting embodiment, examples of such Fc regions includethe Fc regions of human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3(SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8).

Fcγ Receptor (FcγR)

“Fcγ receptor” (also called “FcγR”) refers to a receptor capable ofbinding to the Fc region of monoclonal IgG1, IgG2, IgG3, or IgG4antibodies; and means all members belonging to the family of proteinssubstantially encoded by Fcγ receptor genes. In humans, the familyincludes FcγRI (CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc;FcγRII (CD32) including isoforms FcγRIIa (including allotype H131 andR131, i.e., FcγRIIa(H) and FcγRIIa(R)), FcγRIIb (including FcγRIIb-1 andFcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including isoform FcγRIIIa(including allotype V158 and F158, i.e., FcγRIIIa(V) and FcγRIIIa(F))and FcγRIIIb (including allotype FcγRIIIb-NA1 and FcγRIIIb-NA2); as wellas all unidentified human FcγRs, FcγR isoforms, and allotypes thereof;but the family is not limited to these examples. Without being limitedthereto, FcγRs include those derived from humans, mice, rats, rabbits,and monkeys. FcγRs may be derived from any organism. Mouse FcγRs includeFcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (FcγRIV,CD16-2), as well as all unidentified mouse FcγRs, FcγR isoforms, andallotypes thereof, but they are not limited to these examples. Preferredexamples of such Fcγ receptors include, human FcγRI (CD64), FcγRIIa(CD32), FcγRIIb (CD32), FcγRIIIa (CD16), and/or FcγRIIIb (CD16). Thepolynucleotide sequence and amino acid sequence of human FcγRI are shownin SEQ ID NOs: 9 (NM 000566.3) and 10 (NP 000557.1), respectively; thepolynucleotide sequence and amino acid sequence of human FcγRIIa(allotype H131) are shown in SEQ ID NOs: 11 (BCO20823.1) and 12(AAH20823.1), respectively (allotype R131 is a sequence in which theamino acid at position 166 of SEQ ID NO: 12 is substituted with Arg);the polynucleotide sequence and amino acid sequence of FcγIIb are shownin SEQ ID NOs: 13 (BC146678.1) and 14 (AAI46679.1), respectively; thepolynucleotide sequence and amino acid sequence of FcγRIIIa are shown inSEQ ID NOs: 15 (BCO33678.1) and 16 (AAH33678.1), respectively; and thepolynucleotide sequence and amino acid sequence of FcγRIIIb are shown inSEQ ID NOs: 17 (BC128562.1) and 18 (AAI28563.1), respectively (RefSeqaccession number or such is shown in parentheses). Whether an Fcγreceptor has binding activity to the Fc region of a monoclonal IgG1,IgG2, IgG3, or IgG4 antibody can be assessed by ALPHA (AmplifiedLuminescent Proximity Homogeneous Assay) screen, surface plasmonresonance (SPR)-based BIACORE methods, and others (Proc. Natl. Acad.Sci. USA (2006) 103(11), 4005-4010), in addition to the above-describedFACS and ELISA formats.

In FcγRI (CD64) including FcγRIa, FcγRIb, and FcγRIc, and FcγRIII (CD16)including isoforms FcγRIIIa (including allotypes V158 and F158) andFcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), a chainthat binds to the Fc region of IgG is associated with common γ chainhaving ITAM responsible for transduction of intracellular activationsignal. Meanwhile, the cytoplasmic domain of FcγRII (CD32) includingisoforms FcγRIIa (including allotypes H131 and R131) and FcγRIIccontains ITAM. These receptors are expressed on many immune cells suchas macrophages, mast cells, and antigen-presenting cells. The activationsignal transduced upon binding of these receptors to the Fc region ofIgG results in enhancement of the phagocytic activity of macrophages,inflammatory cytokine production, mast cell degranulation, and theenhanced function of antigen-presenting cells. Fcγ receptors having theability to transduce the activation signal as described above are hereinreferred to as activating Fcγ receptors.

Meanwhile, the intracytoplasmic domain of FcγRIIb (including FcγRIIb-1and FcγRIIb-2) contains ITIM responsible for transduction of inhibitorysignals. The crosslinking between FcγRIIb and B cell receptor (BCR) on Bcells suppresses the activation signal from BCR, which results insuppression of antibody production via BCR. The crosslinking of FcγRIIIand FcγRIIb on macrophages suppresses the phagocytic activity andinflammatory cytokine production. Fcγ receptors having the ability totransduce the inhibitory signal as described above are herein referredto as inhibitory Fcγ receptor.

FcγR-Binding Activity of Fc Region

As mentioned above, Fc regions having an Fcγ receptor-binding activityare examples of Fc regions comprised in the antigen-binding molecules ofthe present invention. A non-limiting embodiment of such an Fc regionincludes the Fc region of human IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO:6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8). Whether an Fcγ receptorhas binding activity to the Fc region of a monoclonal IgG1, IgG2, IgG3,or IgG4 antibody can be assessed by ALPHA screen (Amplified LuminescentProximity Homogeneous Assay), surface plasmon resonance (SPR)-basedBIACORE method, and others (Proc. Natl. Acad. Sci. U.S.A. (2006)103(11), 4005-4010), in addition to the above-described FACS and ELISAformats.

ALPHA screen is performed by the ALPHA technology based on the principledescribed below using two types of beads: donor and acceptor beads. Aluminescent signal is detected only when molecules linked to the donorbeads interact biologically with molecules linked to the acceptor beadsand when the two beads are located in close proximity. Excited by laserbeam, the photosensitizer in a donor bead converts oxygen around thebead into excited singlet oxygen. When the singlet oxygen diffusesaround the donor beads and reaches the acceptor beads located in closeproximity, a chemiluminescent reaction within the acceptor beads isinduced. This reaction ultimately results in light emission. Ifmolecules linked to the donor beads do not interact with moleculeslinked to the acceptor beads, the singlet oxygen produced by donor beadsdo not reach the acceptor beads and chemiluminescent reaction does notoccur.

For example, a biotin-labeled antigen-binding molecule comprising Fcregion is immobilized to the donor beads and glutathione S-transferase(GST)-tagged Fcγ receptor is immobilized to the acceptor beads. In theabsence of an antigen-binding molecule comprising a competitive Fcregion variant, Fcγ receptor interacts with an antigen-binding moleculecomprising a native Fc region, inducing a signal of 520 to 620 nm as aresult. The antigen-binding molecule having a non-tagged Fc regionvariant competes with the antigen-binding molecule comprising a nativeFc region for the interaction with Fcγ receptor. The relative bindingaffinity can be determined by quantifying the reduction of fluorescenceas a result of competition. Methods for biotinylating theantigen-binding molecules such as antibodies using Sulfo-NHS-biotin orthe like are known. Appropriate methods for adding the GST tag to an Fcγreceptor include methods that involve fusing polypeptides encoding Fcγand GST in-frame, expressing the fused gene using cells introduced witha vector to which the gene is operablye linked, and then purifying usinga glutathione column. The induced signal can be preferably analyzed, forexample, by fitting to a one-site competition model based on nonlinearregression analysis using software such as GRAPHPAD PRISM (GraphPad; SanDiego).

One of the substances for observing their interaction is immobilized asa ligand onto the gold thin layer of a sensor chip. When light is shedon the rear surface of the sensor chip so that total reflection occursat the interface between the gold thin layer and glass, the intensity ofreflected light is partially reduced at a certain site (SPR signal). Theother substance for observing their interaction is injected as ananalyte onto the surface of the sensor chip. The mass of immobilizedligand molecule increases when the analyte binds to the ligand. Thisalters the refraction index of solvent on the surface of the sensorchip. The change in refraction index causes a positional shift of SPRsignal (conversely, the dissociation shifts the signal back to theoriginal position). In the Biacore system, the amount of shift describedabove (i.e., the change of mass on the sensor chip surface) is plottedon the vertical axis, and thus the change of mass over time is shown asmeasured data (sensorgram). Kinetic parameters (association rateconstant (ka) and dissociation rate constant (kd)) are determined fromthe curve of sensorgram, and affinity (KD) is determined from the ratiobetween these constants. Inhibition assay is preferably used in theBIACORE methods. Examples of such inhibition assay are described inProc. Natl. Acad. Sci. U.S.A. (2006) 103(11), 4005-4010.

Fc Regions with Altered Fcγ Receptor (FcγR) Binding

In addition to the Fc region of human IgG1 (SEQ ID NO: 5), IgG2 (SEQ IDNO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQ ID NO: 8), an Fc region withaltered FcγR binding, which has a higher Fcγ receptor-binding activitythan an Fc region of a native human IgG may be appropriately used as anFc region included in the present invention. Herein, “Fc region of anative human IgG” refers to an Fc region in which the sugar chain bondedto position 297 (EU numbering) of the Fc region of human IgG1, IgG2,IgG3, or IgG4 shown in SEQ ID NOs: 5, 6, 7, or 8 is a fucose-containingsugar chain. Such Fc regions with altered FcγR binding may be producedby altering amino acids of the Fc region of a native human IgG. Whetherthe FcγR-binding activity of an Fc region with altered FcγR binding ishigher than that of an Fc region of a native human IgG can be determinedappropriately using methods described in the abovementioned section onbinding activity.

In the present invention, “alteration of amino acids” or “amino acidalteration” of an Fc region includes alteration into an amino acidsequence which is different from that of the starting Fc region. Thestarting Fc region may be any Fc region, as long as a variant modifiedfrom the starting Fc region can bind to human Fcγ receptor in a neutralpH range. Furthermore, an Fc region altered from a starting Fc regionwhich had been already altered can also be used preferably as an Fcregion of the present invention. The “starting Fc region” can refer tothe polypeptide itself, a composition comprising the starting Fc region,or an amino acid sequence encoding the starting Fc region. Starting Fcregions can comprise known Fc regions produced via recombinationdescribed briefly in the section “Antibodies”. The origin of starting Fcregions is not limited, and they may be obtained from human or anynonhuman organisms. Such organisms preferably include mice, rats, guineapigs, hamsters, gerbils, cats, rabbits, dogs, goats, sheep, bovines,horses, camels and organisms selected from nonhuman primates. In anotherembodiment, starting Fc regions can also be obtained from cynomolgusmonkeys, marmosets, rhesus monkeys, chimpanzees, or humans. Starting Fcregions can be obtained preferably from human IgG1; however, they arenot limited to any particular IgG class. This means that an Fc region ofhuman IgG1, IgG2, IgG3, or IgG4 can be used appropriately as a startingFc region, and herein also means that an Fc region of an arbitrary IgGclass or subclass derived from any organisms described above can bepreferably used as a starting Fc region. Examples of native IgG variantsor altered forms are described in published documents (Curr. Opin.Biotechnol. (2009) 20 (6): 685-91; Curr. Opin. Immunol. (2008) 20 (4),460-470; Protein Eng. Des. Sel. (2010) 23 (4): 195-202; InternationalPublication Nos. WO 2009/086320, WO 2008/092117, WO 2007/041635, and WO2006/105338); however, they are not limited to the examples.

Examples of alterations include those with one or more mutations, forexample, mutations by substitution of different amino acid residues foramino acids of starting Fc regions, by insertion of one or more aminoacid residues into starting Fc regions, or by deletion of one or moreamino acids from starting Fc region. Preferably, the amino acidsequences of altered Fc regions comprise at least a part of the aminoacid sequence of a non-native Fc region. Such variants necessarily havesequence identity or similarity less than 100% to their starting Fcregion. In a preferred embodiment, the variants have amino acid sequenceidentity or similarity about 75% to less than 100%, more preferablyabout 80% to less than 100%, even more preferably about 85% to less than100%, still more preferably about 90% to less than 100%, and yet morepreferably about 95% to less than 100% to the amino acid sequence oftheir starting Fc region. In a non-limiting embodiment of the presentinvention, at least one amino acid is different between an FcγR-bindingaltered Fc region of the present invention and its starting Fc region.Amino acid difference between an FcγR-binding altered Fc region of thepresent invention and its starting Fc region can also be preferablyspecified based on the specific amino acid differences at theabove-described specific amino acid positions by EU numbering. Examplesof methods of preparing such variants are shown in the section“Alteration of amino acids”.

Included in the antigen-binding molecules of the present invention, anFc region with altered FcγR binding, which has a higher Fcγreceptor-binding activity than that of an Fc region of a native humanIgG, (an FcγR binding-altered Fc region) may be obtained by any method.Specifically, the Fc region with altered FcγR binding may be obtained byaltering amino acids of an IgG-type human immunoglobulin used as astarting Fc region. Preferred Fc regions of the IgG-type immunoglobulinsfor alteration include, for example, those of human IgGs shown in SEQ IDNOs: 5, 6, 7, or 8 (IgG1, IgG2, IgG3, or IgG4, respectively, andvariants thereof).

Amino acids of any positions may be altered into other amino acids, aslong as the binding activity toward the Fcγ receptor is higher than thatof the Fc region of a native human IgG. When the antigen-bindingmolecule contains a human IgG1 Fc region as the human Fc region, itpreferably contains an alteration that yields the effect of a higher Fcγreceptor-binding activity than that of the Fc region of a native humanIgG, in which the sugar chain bound at position 297 (EU numbering) is afucose-containing sugar chain. Such amino acid alterations have beenreported, for example, in international publications such asWO2007/024249, WO2007/021841, WO2006/031370, WO2000/042072,WO2004/029207, WO2004/099249, WO2006/105338, WO2007/041635,WO2008/092117, WO2005/070963, WO2006/020114, WO2006/116260, andWO2006/023403.

Examples of such amino acids that may be altered include at least one ormore amino acids selected from the group consisting of positions 221,222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255,256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,304, 305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327,328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378,379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 (EUnumbering). An Fc region (Fc region with altered FcγR binding) having ahigher Fcγ receptor-binding activity than that of an Fc region of anative human IgG can be obtained by altering these amino acids.

Examples of particularly preferable alterations for use in the presentinvention include at least one or more amino acid alterations selectedfrom the group consisting of:

Lys or Tyr for the amino acid of position 221;Phe, Trp, Glu, or Tyr for the amino acid of position 222;Phe, Trp, Glu, or Lys for the amino acid of position 223;Phe, Trp, Glu, or Tyr for the amino acid of position 224;Glu, Lys, or Trp for the amino acid of position 225;Glu, Gly, Lys, or Tyr for the amino acid of position 227;Glu, Gly, Lys, or Tyr for the amino acid of position 228;Ala, Glu, Gly, or Tyr for the amino acid of position 230;Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 231;Glu, Gly, Lys, or Tyr for the amino acid of position 232;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid of position 233;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid of position 234;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid of position 235;Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid of position 236;Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid of position 237;Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid of position 238;Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,Val, Trp, or Tyr for the amino acid of position 239;Ala, Ile, Met, or Thr for the amino acid of position 240;Asp, Glu, Leu, Arg, Trp, or Tyr for the amino acid of position 241;Leu, Glu, Leu, Gln, Arg, Trp, or Tyr for the amino acid of position 243;His for the amino acid of position 244;Ala for the amino acid of position 245;Asp, Glu, His, or Tyr for the amino acid of position 246;Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acidof position 247;Glu, His, Gln, or Tyr for the amino acid of position 249;Glu or Gln for the amino acid of position 250;Phe for the amino acid of position 251;Phe, Met, or Tyr for the amino acid of position 254;Glu, Leu, or Tyr for the amino acid of position 255;Ala, Met, or Pro for the amino acid of position 256;Asp, Glu, His, Ser, or Tyr for the amino acid of position 258;Asp, Glu, His, or Tyr for the amino acid of position 260;Ala, Glu, Phe, Ile, or Thr for the amino acid of position 262;Ala, Ile, Met, or Thr for the amino acid of position 263;Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Trp, or Tyr for the amino acid of position 264;Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid of position 265;Ala, Ile, Met, or Thr for the amino acid of position 266;Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val,Trp, or Tyr for the amino acid of position 267;Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trpfor the amino acid of position 268;Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, orTyr for the amino acid of position 269;Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyrfor the amino acid of position 270;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid of position 271;Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, orTyr for the amino acid of position 272;Phe or Ile for the amino acid of position 273;Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, or Tyr for the amino acid of position 274;Leu or Trp for the amino acid of position 275;Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, orTyr for the amino acid of position 276;Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, or Trp for the amino acid of position 278;Ala for the amino acid of position 279;Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, or Tyr for the amino acid ofposition 280;Asp, Lys, Pro, or Tyr for the amino acid of position 281;Glu, Gly, Lys, Pro, or Tyr for the amino acid of position 282;Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr for the amino acidof position 283;Asp, Glu, Leu, Asn, Thr, or Tyr for the amino acid of position 284;Asp, Glu, Lys, Gln, Trp, or Tyr for the amino acid of position 285;Glu, Gly, Pro, or Tyr for the amino acid of position 286;Asn, Asp, Glu, or Tyr for the amino acid of position 288;Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for the amino acid ofposition 290;Asp, Glu, Gly, His, Ile, Gln, or Thr for the amino acid of position 291;Ala, Asp, Glu, Pro, Thr, or Tyr for the amino acid of position 292;Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyrfor the amino acid of position 293;Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, orTyr for the amino acid of position 294;Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val,Trp, or Tyr for the amino acid of position 295;Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, orVal for the amino acid of position 296;Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid of position 297;Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, orTyr for the amino acid of position 298;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Val, Trp, or Tyr for the amino acid of position 299;Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, or Trp for the amino acid of position 300;Asp, Glu, His, or Tyr for the amino acid of position 301;Ile for the amino acid of position 302;Asp, Gly, or Tyr for the amino acid of position 303;Asp, His, Leu, Asn, or Thr for the amino acid of position 304;Glu, Ile, Thr, or Tyr for the amino acid of position 305;Ala, Asp, Asn, Thr, Val, or Tyr for the amino acid of position 311;Phe for the amino acid of position 313;Leu for the amino acid of position 315;Glu or Gln for the amino acid of position 317;His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr for the amino acid ofposition 318;Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr forthe amino acid of position 320;Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, or Tyr for theamino acid of position 322;Ile for the amino acid of position 323;Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr forthe amino acid of position 324;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid of position 325;Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, orTyr for the amino acid of position 326;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr,Val, Trp, or Tyr for the amino acid of position 327;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid of position 328;Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid of position 329;Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid of position 330;Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr for theamino acid of position 331;Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid of position 332;Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyrfor the amino acid of position 333;Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of position 334;Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyrfor the amino acid of position 335;Glu, Lys, or Tyr for the amino acid of position 336;Glu, His, or Asn for the amino acid of position 337;Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr for the aminoacid of position 339;Ala or Val for the amino acid of position 376;Gly or Lys for the amino acid of position 377;Asp for the amino acid of position 378;Asn for the amino acid of position 379;Ala, Asn, or Ser for the amino acid of position 380;Ala or Ile for the amino acid of position 382;Glu for the amino acid of position 385;Thr for the amino acid of position 392;Leu for the amino acid of position 396;Lys for the amino acid of position 421;Asn for the amino acid of position 427;Phe or Leu for the amino acid of position 428;Met for the amino acid of position 429;Trp for the amino acid of position 434;Ile for the amino acid of position 436; andGly, His, Ile, Leu, or Tyr for the amino acid of position 440;as indicated by EU numbering in the Fc region. The number of amino acidsto be altered is not particularly limited; and amino acid may be alteredat only one site or amino acids may be altered at two or more sites.Examples of combinations for amino acid alterations at two or more sitesinclude those described in Table 1 (Tables 1-1 to 1-3).

TABLE 1-1 Combination of amino acids Combination of amino acidsK370E/P396L/D270E S239Q/I332Q Q419H/P396L/D270E S267D/I332EV240A/P396L/D270E S267E/I332E R255L/P396L/D270E S267L/A327SR255L/P396L/D270E S267Q/A327S R255L/P396L/D270E/R292G S298A/I332ER255L/P396L/D270E S304T/I332E R255L/P396L/D270E/Y300L S324G/I332DF243L/D270E/K392N/P396L S324G/I332E F243L/R255L/D270E/P396L S324I/I332DF243L/R292P/Y300L/V305I/P396L S324I/I332E F243L/R292P/Y300L/P396LT260H/I332E F243L/R292P/Y300L T335D/I332E F243L/R292P/P396L V240I/V266IF243L/R292P/V305I V264I/I332E F243L/R292P D265F/N297E/I332ES298A/E333A/K334A D265Y/N297D/I332E E380A/T307A F243L/V262I/V264WK326M/E333S N297D/A330Y/I332E K326A/E333A N297D/T299E/I332E S3I7A/K353AN297D/T299F/I332E A327D/I332E N297D/T299H/I332E A330L/I332EN297D/T299I/I332E A330Y/I332E N297D/T299L/I332E E258H/I332EN297D/T299V/I332E E272H/I332E P230A/E233D/I332E E272I/N276DP244H/P245A/P247V E272R/I332E S239D/A330L/I332E E283H/I332ES239D/A330Y/I332E E293R/I332E S239D/H268E/A330Y F241L/V262IS239D/I332E/A327A F241W/F243W S239D/I332E/A330ITable 1-2 is a continuation of Table 1-1.

TABLE 1-2 F243L/V264I S239D/N297D/I332E H268D/A330Y S239D/S298A/I332EH268E/A330Y S239D/V264I/I332E K246H/I332E S239E/N297D/I332E L234D/I332ES239E/V264I/I332E L234E/I332E S239N/A330L/I232E L234G/I332ES239N/A330Y/I332E L234I/I332E S239N/S298A/I332E L234I/L235DS239Q/V2641/I332E L234Y/I332E V264E/N297D/I332E L235D/I332EV264I/A330L/I332E L235E/I332E V264I/A330Y/I332E L235I/I332EV264I/S298A/I332E L235S/I332E Y296D/N297D/I332E L328A/I332DY296E/N297D/I332E L328D/I332D Y296H/N297D/I332E L328D/I332EY296N/N297D/I332E L328E/I332D Y296Q/N297D/I332E L328E/I332EY296T/N297D/I332E L328F/I332D D265Y/N297D/T299L/I332E L328F/I332EF241E/F243Q/V262T/V264E L328H/I332E F241E/F243R/V262E/V264R L328I/I332DF241E/F243Y/V262T/V264R L328I/I332E F241L/F243L/V262I/V264I L328M/I332DF241R/F243Q/V262T/V264R L328M/I332E F241S/F243H/V262T/V264T I328N/I332DF241W/F243W/V262A/V264A L328N/I332E F241Y/F243Y/V626T/V264T L328Q/I332DI332E/A330Y/H268E/A327A L328Q/I332E N297D/I332E/S239D/A330L L328T/I332DN297D/S298A/A330Y/I332E L328T/I332E S239D/A330Y/I332E/K326E L328V/I332DS239D/A330Y/I332E/K326T L328V/I332E S239D/A330Y/I332E/L234I L328Y/I332DS239D/A330Y/I332E/L235DTable 1-3 is a continuation of Table 1-2.

TABLE 1-3 L328Y/I332E S239D/A330Y/I332E/V240I N297D/I332ES239D/A330Y/I332E/V264T N297E/I332E S239D/A330Y/I332E/V266I N297S/I332ES239D/D265F/N297D/I322E P227G/I332E S239D/D265H/N297D/I332E P230A/E233DS239D/D265I/N297D/I332E Q295E/I332E S239D/D265L/N297D/I332E R255Y/I332ES239D/D265T/N297D/I332E S239D/I332D S239D/D265V/N297D/I332E S239D/I332ES239D/D265Y/N297D/I332E S239D/I332N S239D/I332E/A330Y/A327A S239D/I332QS239D/I332E/H268E/A327A S239E/D265G S239D/I332E/H268E/A330Y S239E/D265NS239D/N297D/I332E/A330Y S239E/D265Q S239D/N297D/I332E/K326E S239E/I332DS239D/N297D/I332E/L235D S239E/I332E S239D/V264I/A330L/I332E S239E/I332NS239D/V264I/S298A/I332E S239E/I332Q S239E/V264I/A330Y/I332E S239N/I332DF241E/F243Q/V262T/V264E/I332E S239N/I332E F241E/F243R/V262E/V264R/I332ES239N/I332N F241E/F243Y/V262T/V264R/I332E S239N/I332QF241R/F243Q/V262T/V264R/I332E S239Q/I332D S239D/I332E/H268E/A330Y/A327AS239Q/I332E S239E/V264I/S298A/A330Y/I332E S239Q/I332NF241Y/F243Y/V262T/V264T/N297D/I332E S267E/L328F G236D/S267E S239D/S267E

For the pH conditions to measure the binding activity of the Fcγreceptor binding domain and the Fcγ receptor contained in theantigen-binding molecule of the present invention, conditions in anacidic pH range or in a neutral pH range may be suitably used. Theacidic pH range or neutral pH range, as a condition to measure thebinding activity of the Fcγ receptor binding domain and the Fcγ receptorcontained in the antigen-binding molecule of the present invention,generally indicates pH 5.8 to pH 8.0. Preferably, it is a rangeindicated with arbitrary pH values between pH 6.0 and pH 7.4; andpreferably, it is selected from pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4,pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3,and pH 7.4; and particularly preferably, it is pH 6.15 to 7.4, which isclose to the pH of cancer tissues (Vaupel et al., Cancer Res. (1989) 49,6449-6665). With regard to the temperature used as a measurementcondition, the binding affinity between an Fcγ receptor binding domainand a human Fcγ receptor can be evaluated at any temperature between 10°C. and 50° C. Preferably, a temperature between 15° C. and 40° C. isused to determine the binding affinity between a human Fcγ receptorbinding domain and Fcγ receptor. More preferably, any temperaturebetween 20° C. and 35° C., such as any single temperature from 20° C.,21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C.,30° C., 31° C., 32° C., 33° C., 34° C., and 35° C., can be similarlyused to determine the binding affinity between an Fcγ receptor bindingdomain and an Fcγ receptor. A temperature of 25° C. is a non-limitingexample in an embodiment of the present invention.

Herein, “Fe region with altered FcγR binding has a higher Fcγreceptor-binding activity than the native Fc region” means that thehuman Fcγ receptor-binding activity of the Fc region with altered FcγRbinding toward any of the human Fcγ receptors of FcγRI, FcγRIIa,FcγRIIb, FcγRIIIa, and/or FcγRIIIb is higher than the binding activityof the native Fc region toward these human Fcγ receptors. For example,it means that based on an above-described analytical method, incomparison to the binding activity of an antigen-binding moleculecontaining a native human IgG Fc region as a control, the bindingactivity of the antigen-binding molecule comprising an Fc region withaltered FcγR binding is 105% or more, preferably 110% or more, 115% ormore, 120% or more, 125% or more, particularly preferably 130% or more,135% or more, 140% or more, 145% or more, 150% or more, 155% or more,160% or more, 165% or more, 170% or more, 175% or more, 180% or more,185% or more, 190% or more, 195% or more, 2-fold or more, 2.5-fold ormore, 3-fold or more, 3.5-fold or more, 4-fold or more, 4.5-fold ormore, 5-fold or more, 7.5-fold or more, 10-fold or more, 20-fold ormore, 30-fold or more, 40-fold or more, 50-fold or more, 60-fold ormore, 70-fold or more, 80-fold or more, 90-fold or more, or 100-fold ormore. The starting Fc region may be used as a native Fc region, andnative Fc regions of antibodies of the same subclass may also be used.

In the present invention, an Fc region of a native human IgG in whichthe sugar chain bonded to the amino acid at position 297 (EU numbering)is a fucose-containing sugar chain, is suitably used as a native Fcregion of human IgG to be used as a control. Whether or not the sugarchain bonded to the amino acid at position 297 (EU numbering) is afucose-containing sugar chain can be determined using the techniquedescribed in Non-Patent Document 6. For example, it is possible todetermine whether or not the sugar chain bonded to the native human IgGFc region is a fucose-containing sugar chain by a method such as the onebelow. Sugar chain is dissociated from a native human IgG to be tested,by reacting the test native human IgG with N-Glycosidase F (Rochediagnostics) (Weitzhandler et al. (J. Pharma. Sciences (1994) 83, 12,1670-1675)). Next, a dried concentrate of a reaction solution from whichprotein has been removed by reaction with ethanol (Schenk et al. (J.Clin. Investigation (2001) 108 (11) 1687-1695)) is fluorescently labeledwith 2-aminopyridine (Bigge et al. (Anal. Biochem. (1995) 230 (2)229-238)). Reagents are removed by solid extraction using a cellulosecartridge, and the fluorescently labeled 2-AB-modified sugar chain isanalyzed by normal-phase chromatography. It is possible to determinewhether or not the sugar chain bonded to the native Fc region of a humanIgG is a fucose-containing sugar chain by observing the detectedchromatogram peaks.

As an antigen-binding molecule containing a native Fc region of anantibody of the same subclass, which is to be used as a control, anantigen-binding molecule having an Fc region of a monoclonal IgGantibody may be suitably used. The structures of the Fc regions aredescribed in SEQ ID NO: 5 (A is added to the N terminus of DatabaseAccession No. AAC82527.1), SEQ ID NO: 6 (A is added to the N terminus ofDatabase Accession No. AAB59393.1), SEQ ID NO: 7 (Database Accession No.CAA27268.1), and SEQ ID NO: 8 (A is added to the N terminus of DatabaseAccession No. AAB59394.1). Further, when an antigen-binding moleculecontaining an Fc region of a particular antibody isotype is used as thetest substance, the effect of the antigen-binding molecule containingthe test Fc region on Fcγ receptor-binding activity is tested by usingas a control an antigen-binding molecule having an Fc region of amonoclonal IgG antibody of that particular isotype. In this way,antigen-binding molecules containing an Fc region of which Fcγreceptor-binding activity is demonstrated to be high are suitablyselected.

Fc Regions Having a Selective Binding Activity Toward an Fcγ Receptor

Examples of Fcγ receptor binding domains suitable for use in the presentinvention include Fcγ receptor binding domains having a higher bindingactivity to a particular Fcγ receptor than to other Fcγ receptors (Fcγreceptor binding domains having a selective binding activity to an Fcγreceptor). When an antibody is used as the antigen-binding molecule(when an Fc region is used as the Fcγ receptor binding domain), a singleantibody molecule can only bind to a single Fcγ receptor molecule.Therefore, a single antigen-binding molecule cannot bind to otheractivating FcγRs in an inhibitory Fcγ receptor-bound state, and cannotbind to other activating Fcγ receptors or inhibitory Fcγ receptors in anactivating Fcγ receptor-bound state.

Fc Regions with a Higher Binding Activity Toward an Activating FcγReceptor than the Binding Activity Toward an Inhibitory Fcγ Receptor

As described above, preferable activating Fcγ receptors include FcγRI(CD64) including FcγRIa, FcγRIb, and FcγRIc; FcγRIIa; and FcγRIII (CD16)including FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb(including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2). Meanwhile,preferred examples of inhibitory Fcγ receptors include FcγRIIb(including FcγRIIb-1 and FcγRIIb-2).

Herein, an example of a case where the binding activity toward a certainFcγ receptor is higher than the binding activity toward another Fcγreceptor is the case where the binding activity toward an activating Fcγreceptor is higher than the binding activity toward an inhibitory Fcγreceptor. In this case, the binding activity of the Fc region toward anyof the human Fcγ receptors of FcγRIa, FcγRIIa, FcγRIIIa, and/or FcγRIIIbis said to be higher than the binding activity toward FcγRIIb. Forexample, this means that, based on an above-described analytical method,the binding activity of an antigen-binding molecule containing the Fcregion toward any of the human Fcγ receptors, FcγRIa, FcγRIIa, FcγRIIIa,and/or FcγRIIIb, is 105% or more, preferably 110% or more, 120% or more,130% or more, 140% or more, particularly preferably 150% or more, 160%or more, 170% or more, 180% or more, 190% or more, 200% or more, 250% ormore, 300% or more, 350% or more, 400% or more, 450% or more, 500% ormore, 750% or more, 10-fold or more, 20-fold or more, 30-fold or more,40-fold or more, 50-fold or more, 60-fold, 70-fold, 80-fold, 90-fold, or100-fold or more as compared with the binding activity toward FcγRIIb.The Fc region with a higher binding activity toward activating Fcγreceptors than to inhibitory Fcγ receptors may be favorably included inantigen-binding molecules of the present invention whose antigen-bindingdomain binds to a membrane-type molecule. IgG1 antibodies containingsuch Fc regions are known to enhance the ADCC activity mentioned below.Therefore, antigen-binding molecules containing the Fc-region are alsouseful as antigen-binding molecules to be included in the pharmaceuticalcompositions of the present invention.

In a non-limiting embodiment of the present invention, examples of theFc region with a higher binding activity toward activating Fcγ receptorsthan to inhibitory Fcγ receptors (or having a selective binding activitytoward inhibitory Fcγ receptors) preferably include Fc regions in whichat least one or more amino acids selected from the group consisting ofamino acids at positions 221, 222, 223, 224, 225, 227, 228, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246,247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281,282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297,298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421, 427,428, 429, 434, 436, and 440 indicated by EU numbering mentioned above,have been altered to amino acids different from those of the native Fcregion.

In a non-limiting embodiment of the present invention, examples of theFc region with a higher binding activity toward activating Fcγ receptorsthan to inhibitory Fcγ receptors (or having a selective binding activitytoward inhibitory Fcγ receptors) preferably include Fc regions in whichmultiple amino acids indicated in Tables 1-1 to 1-3 have been altered toamino acids different from those of the native Fc region.

Fc Regions Whose Binding Activity Toward an Inhibitory Fcγ Receptor isHigher than the Binding Activity Toward an Activating Fcγ Receptor

Herein, an example of a case where the binding activity toward a certainFcγ receptor is higher than the binding activity toward another Fcγreceptor is the case where the binding activity toward an inhibitory Fcγreceptor is higher than the binding activity toward an activating Fcγreceptor. In this case, the binding activity of the Fc region towardFcγRIIb is said to be higher than the binding activity toward any of thehuman Fcγ receptors of FcγRIa, FcγRIIa, FcγRIIIa, and/or FcγRIIIb. Forexample, this means that, based on an above-described analytical method,the binding activity of an antigen-binding molecule containing the Fcregion toward FcγRIIb is 105% or more, preferably 110% or more, 120% ormore, 130% or more, 140% or more, particularly preferably 150% or more,160% or more, 170% or more, 180% or more, 190% or more, 200% or more,250% or more, 300% or more, 350% or more, 400% or more, 450% or more,500% or more, 750% or more, 10-fold or more, 20-fold or more, 30-fold ormore, 40-fold or more, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or100-fold or more as compared with the binding activity toward any of thehuman Fcγ receptors of FcγRIa, FcγRIIa, FcγRIIIa, and/or FcγRIIIb. TheFc region with a higher binding activity toward inhibitory Fcγ receptorsthan to activating Fcγ receptors may be favorably included inantigen-binding molecules of the present invention whose antigen-bindingdomain binds to a soluble molecule.

In a non-limiting embodiment of the present invention, examples of theFc region with a higher binding activity toward inhibitory Fcγ receptorsthan to activating Fcγ receptors (or having a selective binding activitytoward inhibitory Fcγ receptors) preferably include Fc regions in which,of the amino acids of the above Fc region, the amino acids at 238 and328 indicated by EU numbering are altered to amino acids different fromthose of the native Fc region.

In a non-limiting embodiment of the present invention, examples of theFc region with a higher binding activity toward inhibitory Fcγ receptorsthan to activating Fcγ receptors (or having a selective binding activitytoward inhibitory Fcγ receptors) preferably include Fc regions alteredat any one or more of the amino acids in the above Fc region asindicated by EU numbering: the amino acid at position 238 (indicated byEU numbering) is altered into Asp; and the amino acid at position 328(indicated by EU numbering) is altered into Glu. Furthermore, as the Fcregions having a selective binding activity toward inhibitory Fcγreceptors, the Fc regions or alterations described in US 2009/0136485can be suitably selected.

In another non-limiting embodiment of the present invention, preferredexamples include Fc regions altered at any one or more of the aminoacids in the above Fc region as indicated by EU numbering: the aminoacid at position 238 (indicated by EU numbering) to Asp; and the aminoacid at position 328 (indicated by EU numbering) to Glu.

In still another non-limiting embodiment of the present invention,preferred examples include Fc regions that have one or more of thealterations exemplified in PCT/JP2012/054624: substitution of Pro atposition 238 (indicated by EU numbering) with Asp; alteration of theamino acid at position 237 (indicated by EU numbering) to Trp;alteration of the amino acid at position 237 (indicated by EU numbering)to Phe; alteration of the amino acid at position 267 (indicated by EUnumbering) to Val; alteration of the amino acid at position 267(indicated by EU numbering) to Gln; alteration of the amino acid atposition 268 (indicated by EU numbering) to Asn; alteration of the aminoacid at position 271 (indicated by EU numbering) to Gly; alteration ofthe amino acid at position 326 (indicated by EU numbering) to Leu;alteration of the amino acid at position 326 (indicated by EU numbering)to Gln; alteration of the amino acid at position 326 (indicated by EUnumbering) to Glu; alteration of the amino acid at position 326(indicated by EU numbering) to Met; alteration of the amino acid atposition 239 (indicated by EU numbering) to Asp; alteration of the aminoacid at position 267 (indicated by EU numbering) to Ala; alteration ofthe amino acid at position 234 (indicated by EU numbering) to Trp;alteration of the amino acid at position 234 (indicated by EU numbering)to Tyr; alteration of the amino acid at position 237 (indicated by EUnumbering) to Ala; alteration of the amino acid at position 237(indicated by EU numbering) to Asp; alteration of the amino acid atposition 237 (indicated by EU numbering) to Glu; alteration of the aminoacid at position 237 (indicated by EU numbering) to Leu; alteration ofthe amino acid at position 237 (indicated by EU numbering) to Met;alteration of the amino acid at position 237 (indicated by EU numbering)to Tyr; alteration of the amino acid at position 330 (indicated by EUnumbering) to Lys; alteration of the amino acid at position 330(indicated by EU numbering) to Arg, alteration of the amino acid atposition 233 (indicated by EU numbering) to Asp, alteration of the aminoacid at position 268 (indicated by EU numbering) to Asp, alteration ofthe amino acid at position 268 (indicated by EU numbering) to Glu,alteration of the amino acid at position 326 (indicated by EU numbering)to Asp, alteration of the amino acid at position 326 (indicated by EUnumbering) to Ser, alteration of the amino acid at position 326(indicated by EU numbering) to Thr, alteration of the amino acid atposition 323 (indicated by EU numbering) to Ile, alteration of the aminoacid at position 323 (indicated by EU numbering) to Leu, alteration ofthe amino acid at position 323 (indicated by EU numbering) to Met,alteration of the amino acid at position 296 (indicated by EU numbering)to Asp, alteration of the amino acid at position 326 (indicated by EUnumbering) to Ala, alteration of the amino acid at position 326(indicated by EU numbering) to Asn, and alteration of the amino acid atposition 330 (indicated by EU numbering) to Met.

Fc Regions with Modified Sugar Chains

Fc regions contained in the antigen-binding molecules provided by thepresent invention may include Fc regions that have been modified so thatthe composition of the sugar-chain-attached Fc regions has a highpercentage of fucose-deficient sugar-chain-attached Fc regions, or ahigh percentage of bisecting N-acetylglucosamine-added Fc regions.Removal of fucose residue from N-acetylglucosamine at the reducing endof N-glycoside linkage complex sugar chains bonded to the antibody Fcregion is known to enhance the affinity to FcγRIIIa (Non-Patent Document6). It is known that for IgG1 antibodies containing such Fc regions, theADCC activity mentioned below is enhanced; therefore, antigen-bindingmolecules containing such Fc regions are also useful as antigen-bindingmolecules to be contained in pharmaceutical compositions of the presentinvention. Examples of antibodies with fucose residue removed fromN-acetylglucosamine at the reducing end of N-glycoside linkage complexsugar chains bonded to the antibody Fc regions are antibodies such as:

antibodies modified by glycosylation (for example, WO 1999/054342); andantibodies deficient in fucose attached to sugar chains (for example, WO2000/061739, WO 2002/031140, and WO 2006/067913).

More specifically, to produce antibodies deficient in fucose attached tosugar chains (for example, WO 2000/061739, WO 2002/031140, and WO2006/067913) as another non-limiting embodiment of antibodies withfucose residue removed from N-acetylglucosamine at the reducing end ofN-glycoside linkage complex sugar chains bonded to the antibody Fcregions, host cells having a low ability to add fucose to sugar chainsare produced by altering the activity of forming the sugar chainstructure of the polypeptide to be glycosylated. Antibodies that lackfucose in their sugar chains can be collected from culture of the hostcells by expressing a desired antibody gene in the host cells.Non-limiting suitable examples of the activity to form the sugar chainstructure of a polypeptide include the activity of a transporter or anenzyme selected from the group consisting of fucosyltransferase (EC2.4.1.152), fucose transporter (SLC35C1), GMD(GDP-mannose-4,6-dehydratase) (EC 4.2.1.47), Fx(GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase) (EC 1.1.1.271), andGFPP (GDP-β-L-fucose pyrophosphorylase (EC 2.7.7.30). As long as theseenzymes or transporters can exhibit their activities, their structuresare not necessarily specified. Herein, proteins that can exhibit theseactivities are referred to as “functional proteins”. In a non-limitingembodiment, methods for altering these activities include deletion ofthese activities. To produce host cells deficient in these activities,known methods such as a method for destroying the genes of thesefunctional proteins to make them unable to function may be appropriatelyemployed (for example, WO2000/061739, WO2002/031140, and WO2006/067913).Host cells deficient in such activities can be produced, for example, bya method that destroys the genes of these functional proteins endogenousto CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63mouse myeloma cells, PER cells, PER.C6 cells, HEK293 cells, hybridomacells, or such, so that the genes are unable to function.

Antibodies that have a sugar chain containing bisecting GlcNAc(WO2002/079255, etc.) are known. In a non-limiting embodiment, hostcells for expressing a gene that encodes a functional protein havingGnTIII (β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase)(EC 2.4.1.144) activity or Ga1T (β-1,4-galactosyltransferase) (EC2.4.1.38) activity are produced to prepare antibodies that havebisecting GlcNAc-containing sugar chains. In another suitablenon-limiting embodiment, host cells that co-express, in addition to theaforementioned functional proteins, a gene encoding a functional proteinhaving human ManII (manosidase II) (3.2.1.114) activity, a gene encodinga functional protein having GnTI (β-1,2-acetylglucosaminyltransferase I)(EC 2.4.1.94) activity, a gene encoding a functional protein havingGnTII (β-1,2-acetylglucosaminyltransferase II) (EC 2.4.1.143) activity,a gene encoding a functional protein having ManI (mannosidase) (EC3.2.1.113) activity, and α-1,6-fucosyl transferase (EC 2.4.1.68), areproduced (WO2004/065540).

Antibodies with fucose residue removed from N-acetylglucosamine at thereducing end of N-glycoside linkage complex sugar chains bonded to theantibody Fc regions and antibodies having sugar chains containingbisecting GlcNAc can be produced, respectively, by transfecting anexpression vector containing the antibody gene into host cells with alow ability to add fucose to sugar chains, and into host cells havingthe activity to form bisecting GlcNAc structure-containing sugar chains.Methods for producing these antibodies can be applied to methods forproducing antigen-binding molecules containing altered Fc regions thathave been modified so that the composition of the sugar-chain-attachedFc regions of the present invention has a high percentage offucose-deficient sugar chain-attached Fc regions or a high percentage ofbisecting N-acetylglucosamine-added Fc regions. The composition of thesugar-chain-attached Fc regions contained in the antigen-bindingmolecules of the present invention produced by such production methodscan be assessed by the method described in “Fc regions with altered Fcγreceptor (FcγR) binding” above.

Multispecific Antigen-Binding Molecules or MultiparatopicAntigen-Binding Molecules

An antigen-binding molecule comprising at least two antigen-bindingdomains in which at least one of the antigen-binding domains binds to afirst epitope in an antigen molecule, and at least another one of theantigen-binding domains binds to a second epitope in the antigenmolecule, is called “multispecific antigen-binding molecule” from theviewpoint of its reaction specificity. When two types of antigen-bindingdomains contained in a single antigen-binding molecule allow binding totwo different epitopes by the antigen-binding molecule, this molecule iscalled “bispecific antigen-binding molecule”. When three types ofantigen-binding domains contained in a single antigen-binding moleculeallow binding to three different epitopes by the antigen-bindingmolecule, this antigen-binding molecule is called “trispecificantigen-binding molecule”.

A paratope in the antigen-binding domain that binds to the first epitopein the antigen molecule and a paratope in the antigen-binding domainthat binds to the second epitope which is structurally different fromthe first epitope have different structures. Therefore, anantigen-binding molecule comprising at least two antigen-binding domainsin which at least one of the antigen-binding domains binds to a firstepitope in an antigen molecule, and at least another one of theantigen-binding domains binds to a second epitope in the antigenmolecule, is called “multiparatopic antigen-binding molecule” from theviewpoint of the specificity of its structure. When two types ofantigen-binding domains contained in a single antigen-binding moleculeallow binding to two different epitopes by the antigen-binding molecule,this molecule is called “biparatopic antigen-binding molecule”. Whenthree types of antigen-binding domains contained in a singleantigen-binding molecule allow binding to three different epitopes bythe antigen-binding molecule, this molecule is called “triparatopicantigen-binding molecule”.

Multivalent multispecific or multiparatopic antigen-binding moleculescomprising one or more antigen-binding domains and methods for preparingthem are described in non-patent documents such as Conrath et al., (J.Biol. Chem. (2001) 276 (10) 7346-7350), Muyldermans (Rev. Mol. Biotech.(2001) 74, 277-302), and Kontermann R. E. (2011) Bispecific Antibodies(Springer-Verlag), and in patent documents such as WO1996/034103 andWO1999/023221. Antigen-binding molecules of the present invention can beproduced using multispecific or multiparatopic antigen-bindingmolecules, and their preparation methods described in these documents.

Bispecific Antibodies and Methods for Producing them

In an embodiment, bispecific antibodies and methods for producing themare mentioned below as examples of the aforementioned multispecific ormultiparatopic antigen-binding molecules and methods for preparing them.Bispecific antibodies are antibodies comprising two types of variableregions that bind specifically to different epitopes. IgG-typebispecific antibodies can be secreted from a hybrid hybridoma (quadroma)produced by fusing two types of hybridomas that produce IgG antibodies(Milstein et al., Nature (1983) 305, 537-540).

When a bispecific antibody is produced by using recombination techniquessuch as those described in the above-mentioned section on antibodies,one may adopt a method that introduces genes encoding heavy chainscontaining the two types of variable regions of interest into cells toco-express them. However, even when only the heavy-chain combination isconsidered, such a co-expression method will produce a mixture of (i) acombination of a pair of heavy chains in which one of the heavy chainscontains a variable region that binds to a first epitope and the otherheavy chain contains a variable region that binds to a second epitope,(ii) a combination of a pair of heavy chains which include only heavychains containing a variable region that binds to the first epitope, and(iii) a combination of a pair of heavy chains which include only heavychains containing a variable region that binds to the second epitope,which are present at a molecular ratio of 2:1:1. It is difficult topurify antigen-binding molecules containing the desired combination ofheavy chains from the mixture of three types of heavy chaincombinations.

When producing bispecific antibodies using such recombinationtechniques, bispecific antibodies containing a heteromeric combinationof heavy chains can be preferentially secreted by adding appropriateamino acid substitutions in the CH3 domains constituting the heavychains. Specifically, this method is conducted by substituting an aminoacid having a larger side chain (knob (which means “bulge”)) for anamino acid in the CH3 domain of one of the heavy chains, andsubstituting an amino acid having a smaller side chain (hole (whichmeans “void”)) for an amino acid in the CH3 domain of the other heavychain so that the knob is placed in the hole. This promotes heteromericheavy chain formation and simultaneously inhibits homomeric heavy chainformation (International Publication No. WO 1996027011; Ridgway et al.,Protein Engineering (1996) 9, 617-621; Merchant et al., NatureBiotechnology (1998) 16, 677-681).

Furthermore, there are also known techniques for producing a bispecificantibody by applying methods for controlling polypeptide association, orassociation of polypeptide-formed heteromeric multimers to theassociation between heavy chains. Specifically, methods for controllingheavy chain formation may be employed to produce a bispecific antibody(International Publication No. WO 2006/106905), in which amino acidresidues forming the interface between the heavy chains are altered toinhibit the association between the heavy chains having the samesequence and to allow the formation of heavy chains of differentsequences. Such methods can be used for generating bispecificantibodies.

In a non-limiting embodiment of the present invention, two polypeptidesconstituting an Fc region derived from a bispecific antibody describedabove can be suitably used as an Fc region to be included in theantigen-binding molecule. More specifically, it is preferable to use twopolypeptides that constitute an Fc region, and which comprise Cys forthe amino acid at position 349 and Trp for the amino acid at position366 according to EU numbering in the amino acid sequence of one of thepolypeptides; and Cys for the amino acid at position 356, Ser for theamino acid at position 366, Ala for the amino acid at position 368, andVal for the amino acid at position 407 as indicated by EU numbering inthe amino acid sequence of the other polypeptide.

In another non-limiting embodiment of the present invention, twopolypeptides that constitute an Fc region and which comprise Asp for theamino acid at position 409 according to EU numbering in the amino acidsequence of one of the polypeptides, and Lys for the amino acid atposition 399 according to EU numbering in the amino acid sequence of theother polypeptide, may be suitably used as the Fc region. In the aboveembodiment, the amino acid at position 409 may be Glu instead of Asp,and the amino acid at position 399 may be Arg instead of Lys. Moreover,in addition to the amino acid Lys at position 399, Asp may be suitablybe added as the amino acid at position 360 or Asp may suitably be addedas the amino acid at position 392.

In still another non-limiting embodiment of the present invention, twopolypeptides that constitute an Fc region, and which comprise Glu forthe amino acid at position 370 according to EU numbering in the aminoacid sequence of one of the polypeptides, and Lys for the amino acid atposition 357 according to EU numbering in the amino acid sequence of theother polypeptide, may be suitably used as the Fc region.

In yet another non-limiting embodiment of the present invention, twopolypeptides that constitute an Fc region, and which comprise Glu forthe amino acid at position 439 according to EU numbering in the aminoacid sequence of one of the polypeptides, and Lys for the amino acid atposition 356 according to EU numbering in the amino acid sequence of theother polypeptide, may be suitably used as the Fc region.

In still yet another non-limiting embodiment of the present invention,any of the embodiments indicated below of combinations from the abovemay be suitably used as the Fc region:

(i) two polypeptides that constitute an Fc region, and which compriseAsp for the amino acid at position 409 and Glu for the amino acid atposition 370 according to EU numbering in the amino acid sequence of oneof the polypeptides, and Lys for the amino acid at position 399 and Lysfor the amino acid at position 357 according to EU numbering in theamino acid sequence of the other polypeptide (in this embodiment, theamino acid at position 370 according to EU numbering may be Asp insteadof Glu, and the amino acid Asp at position 392 may be used instead ofthe amino acid Glu at position 370 according to EU numbering);

(ii) two polypeptides that constitute an Fc region, and which compriseAsp for the amino acid at position 409 and Glu for the amino acid atposition 439 according to EU numbering of the amino acid sequence of oneof the polypeptides; and Lys for the amino acid at position 399 and Lysfor the amino acid at position 356 according to EU numbering in theamino acid sequence of the other polypeptide (in this embodiment, theamino acid Asp at position 360, the amino acid Asp at position 392, orthe amino acid Asp at position 439 may be used instead of the amino acidGlu at position 439 according to EU numbering);

(iii) two polypeptides that constitute an Fc region, and which compriseGlu for the amino acid at position 370 and Glu for the amino acid atposition 439 according to EU numbering in the amino acid sequence of oneof the polypeptides, and Lys for the amino acid at position 357 and Lysfor the amino acid at position 356 according to EU numbering in theamino acid sequence of the other polypeptide; or

two polypeptides that constitute an Fc region, and which comprise Aspthe amino acid at position 409, Glu for the amino acid at position 370,and Glu for the amino acid at position 439 according to EU numbering inthe amino acid sequence of one of the polypeptides; and Lys for theamino acid at position 399, Lys for the amino acid at position 357, andLys for the amino acid at position 356 according to EU numbering in theamino acid sequence of the other polypeptide (in this embodiment, theamino acid at position 370 may not be substituted with Glu, andfurthermore, when the amino acid at position 370 is not substituted withGlu, the amino acid at position 439 may be Asp instead of Glu, or theamino acid Asp at position 392 may be used instead of the amino acid Gluat position 439, according to EU numbering).

Further, in another non-limiting embodiment of the present invention, itmay also be suitable to use two polypeptides that constitute an Fcregion, and which comprise Lys for the amino acid at position 356according to EU numbering in the amino acid sequence of one of thepolypeptides, and Arg for the amino acid at position 435 and Glu for theamino acid at position 439 according to EU numbering in the amino acidsequence of the other polypeptide.

In still another non-limiting embodiment of the present invention, itmay also be suitable to use two polypeptides that constitute an Fcregion and which comprise Lys for the amino acid at position 356 and Lysfor the amino acid at position 357 according to EU numbering in theamino acid sequence of one of the polypeptides, and Glu for the aminoacid at position 370, Arg for the amino acid at position 435, and Glufor the amino acid at position 439 according to EU numbering in theamino acid sequence of the other polypeptide.

Furthermore, in addition to the above-mentioned technologies ofassociating heterologous heavy chains, CrossMab technology which isknown as a technology for associating heterologous light chains, inwhich a light chain forming a variable region that binds to a firstepitope and a light chain forming a variable region that binds to asecond epitope are respectively associated with a heavy chain forming avariable region that binds to the first epitope and a heavy chainforming a variable region that binds to the second epitope (Scaefer etal. (Proc. Natl. Acad. Sci. U.S.A. (2011) 108, 11187-11192)), may alsobe used to produce the multispecific or multiparatopic antigen-bindingmolecules provided by the present invention. Furthermore, Fab-ArmExchange which is known as a technology for associating heterologousheavy chains, in which a heavy chain forming a variable region thatbinds to a first epitope and a heavy chain forming a variable regionthat binds to a second epitope by utilizing that heterologous IgG4 heavychains exchange each other (Labrijn et al. (Proc. Natl. Acad. Sci.U.S.A. (2013) 110, 5145-5150), WO2008119353), may also be used toproduce the multispecific or multiparatopic antigen-binding moleculesprovided by the present invention.

Effector Cells

In the present invention, the term “effector cells” may be used in thebroadest sense including T cells (CD4⁺ (helper lymphocyte) T cellsand/or CD8⁺ (cytotoxic) T cells), multinuclear leucocytes (neutrophils,eosinophils, basophils, mast cells), monocytes, macrophages,histiocytes, or leukocytes such as natural killer cells (NK cells),NK-like T cells, Kupffer cells, Langerhans cells, orlymphokine-activated killer cells (LAK cells), B-lymphocytes, orantigen-presenting cells such as dendritic cells or macrophages.Preferred examples of effector cells include CD8⁺ (cytotoxic) T cells,NK cells, or macrophages. Membrane-type molecules expressed on the cellmembrane of effector cells may be used as antigens to which at least oneantigen-binding domain contained in the antigen-binding molecule of thepresent invention binds. Non-limiting examples of a preferredmembrane-type molecule may be CD3, CD2, CD28, CD44, CD16, CD32, CD64, orNKG2D, NK cell-activating ligands, or polypeptides constituting TCR.

Cytotoxic Substances

In order for antigen-binding molecules of the present invention to bindto cancer cells and exhibit cytotoxic activity, cytotoxic substances maybe linked to antigen-binding molecules. The cytotoxic substances may bechemotherapeutic agents exemplified below, or compounds disclosed inCurr Opin Chem Biol (2010) 14, 529-37 and WO 2009/140242; and thesecompounds are linked to antigen-binding molecules by appropriate linkersand such. When antigen-binding molecules of the present invention areused as pharmaceutical compositions, these cytotoxic substances may belinked to the antigen-binding molecules prior to administration, or theymay be administered before, after, or at the same time when theantigen-binding molecules are administered to subjects (testindividuals, patients, and such).

The later-described modified antigen-binding molecules to whichcytotoxic substances such as chemotherapeutic agents, toxic peptides, orradioactive chemical substances have been linked may also be usedpreferably as antigen-binding molecules of the present invention havingcytotoxic activity. Such modified antigen-binding molecules (hereinafterreferred to as antigen-binding molecule-drug conjugate) can be obtainedby chemically modifying the obtained antigen-binding molecules. Methodsthat have been already established in the field of antibody-drugconjugates and such may be used appropriately as methods for modifyingantigen-binding molecules. Furthermore, a modified antigen-bindingmolecule to which a toxic peptide is linked can be obtained byexpressing in appropriate host cells a fused gene produced by linking agene encoding the toxic peptide in frame with a gene encoding anantigen-binding molecule of the present invention, and then isolating itfrom the cell culture.

Examples of chemotherapeutic agents linked to the antigen-bindingmolecules of the present invention may include:

azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, camptothecin, 10-hydroxycamptothecin,carmustine, celebrex, chlorambucil, cisplatin, irinotecan, carboplatin,cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel,dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone,diethylstilbestrol, doxorubicin, doxorubicin glucuronide, epirubicin,ethinyl estradiol, estramustine, etoposide, etoposide glucuronide,floxuridine, fludarabine, flutamide, fluorouracil, fluoxymesterone,gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin,ifosfamide, leucovorin, lomustine, maytansinoid, mechlorethamine,medroxyprogesterone acetate, megestrol acetate, melphalan,mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenylbutyrate, prednisone, procarbazine, paclitaxel,pentostatin, semustine, streptozocin, tamoxifen, taxanes, taxol,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vinblastine, vinorelbine, and vincristine.

In the present invention, preferred chemotherapeutic agents arelow-molecular-weight chemotherapeutic agents. Low-molecular-weightchemotherapeutic agents are unlikely to interfere with the function ofantigen-binding molecules even after they bind to antigen-bindingmolecules of the present invention. In the present invention,low-molecular-weight chemotherapeutic agents usually have a molecularweight of 100 to 2000, preferably 200 to 1000. The chemotherapeuticagents exemplified herein are all low-molecular-weight chemotherapeuticagents. The chemotherapeutic agents of the present invention includeprodrugs that are converted into active chemotherapeutic agents in vivo.Prodrug activation may be enzymatic conversion or non-enzymaticconversion.

Moreover, cytotoxic substances that are linked to antigen-bindingmolecules of the present invention include, for example, toxic peptides(toxins) such as Pseudomonas exotoxin A, Saporin-s6, Diphtheria toxin,Cnidarian toxin; radioiodine; and photosensitizers. Suitable examples ofthe toxic peptides include the following:

Diphtheria toxin A Chain (Langone et al. (Methods in Enzymology (1983)93, 307-308)); Pseudomonas Exotoxin (Nature Medicine (1996) 2, 350-353);Ricin Chain (Ricin A Chain) (Fulton et al. (J. Biol. Chem. (1986) 261,5314-5319), Sivam et al. (Cancer Res. (1987) 47, 3169-3173), Cumber etal. (J. Immunol. Methods (1990) 135, 15-24), Wawrzynczak et al. (CancerRes. (1990) 50, 7519-7562), and Gheeite et al. (J. Immunol. Methods(1991) 142, 223-230));

Deglicosylated Ricin A Chain (Thorpe et al. (Cancer Res. (1987) 47,5924-5931));

Abrin A Chain (Wawrzynczak et al. (Br. J. Cancer (1992) 66, 361-366),Wawrzynczak et al. (Cancer Res. (1990) 50, 7519-7562), Sivam et al.(Cancer Res. (1987) 47, 3169-3173), and Thorpe et al. (Cancer Res.(1987) 47, 5924-5931));Gelonin (Sivam et al. (Cancer Res. (1987) 47, 3169-3173), Cumber et al.(J. Immunol. Methods (1990) 135, 15-24), Wawrzynczak et al. (CancerRes., (1990) 50, 7519-7562), and Bolognesi et al. (Clin. exp. Immunol.(1992) 89, 341-346));PAP-s; Pokeweed anti-viral protein from seeds (Bolognesi et al. (Clin.exp. Immunol. (1992) 89, 341-346));Briodin (Bolognesi et al. (Clin. exp. Immunol. (1992) 89, 341-346));Saporin (Bolognesi et al. (Clin. exp. Immunol. (1992) 89, 341-346));Momordin (Cumber et al. (J. Immunol. Methods (1990) 135, 15-24);Wawrzynczak et al. (Cancer Res. (1990) 50, 7519-7562); and Bolognesi etal. (Clin. exp. Immunol. (1992) 89, 341-346));Momorcochin (Bolognesi et al. (Clin. exp. Immunol. (1992) 89, 341-346));Dianthin 32 (Bolognesi et al. (Clin. exp. Immunol. (1992) 89, 341-346));Dianthin 30 (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Modeccin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Viscumin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Volkesin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Dodecandrin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Tritin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8));Luffin (Stirpe F., Barbieri L. (FEBS letter (1986) 195, 1-8)); andTrichokirin (Casellas et al. (Eur. J. Biochem. (1988) 176, 581-588), andBolognesi et al. (Clin. exp. Immunol., (1992) 89, 341-346)).

Antigen-Binding Molecule

In the present invention, “an antigen-binding molecule comprising anantigen-binding domain whose antigen-binding activity in the presence ofa target tissue-specific compound is higher than in the absence of thetarget tissue-specific compound” is used in the broadest sense; andspecifically, it includes various types of molecules as long as theyshow antigen-binding activity. Molecules in which an antigen-bindingdomain is linked to an Fc region include, for example, antibodies.Antibodies may include single monoclonal antibodies (including agonisticantibodies and antagonistic antibodies), human antibodies, humanizedantibodies, chimeric antibodies, and such. Alternatively, when used asantibody fragments, they preferably include antigen-binding domains andantigen-binding fragments (for example, Fab, F(ab′)2, scFv, and Fv).Scaffold molecules where three dimensional structures, such asalready-known stable α/β barrel protein structure, are used as ascaffold (base) and only some portions of the structures are made intolibraries to construct antigen-binding domains are also included inantigen-binding molecules of the present invention.

An antigen-binding molecule of the present invention may contain atleast some portions of an Fc region that mediates the binding to Fcγreceptor and/or FcRn. In a non-limiting embodiment, the antigen-bindingmolecule includes, for example, antibodies and Fc fusion proteins. Afusion protein refers to a chimeric polypeptide comprising a polypeptidehaving a first amino acid sequence that is linked to a polypeptidehaving a second amino acid sequence that would not naturally link innature. For example, a fusion protein may comprise a polypeptidecomprising the amino acid sequence of at least a portion of an Fc region(for example, a portion of an Fc region responsible for the binding toFcγ receptor, and/or a portion of an Fc region responsible for thebinding to FcRn). The amino acid sequences may be present in separateproteins that are transported together to a fusion protein, or generallymay be present in a single protein; however, they are included in a newrearrangement in a fusion polypeptide. Fusion proteins can be produced,for example, by chemical synthesis, or by genetic recombinationtechniques to express a polynucleotide encoding peptide regions in adesired arrangement.

Respective domains of the present invention can be linked together vialinkers or directly via polypeptide binding. The linkers comprisearbitrary peptide linkers that can be introduced by genetic engineering,synthetic linkers, and linkers disclosed in, for example, Holliger etal., Protein Engineering (1996) 9(3), 299-305. However, peptide linkersare preferred in the present invention. The length of the peptidelinkers is not particularly limited, and can be suitably selected bythose skilled in the art according to the purpose. The length ispreferably five amino acids or more (without particular limitation, theupper limit is generally 30 amino acids or less, preferably 20 aminoacids or less), and particularly preferably 15 amino acids.

For example, such peptide linkers preferably include:

Ser Gly•Ser Gly•Gly•Ser Ser•Gly•Gly (SEQ ID NO: 19) Gly•Gly•Gly•Ser(SEQ ID NO: 20) Se•Gly•Gly•Gly (SEQ ID NO: 21) Gly•Gly•Gly•Gly•Ser(SEQ ID NO: 22) Se•Gly•Gly•Gly•Gly (SEQ ID NO: 23)Gly•Gly•Gly•Gly•Gly•Ser (SEQ ID NO: 24) Se•Gly•Gly•Gly•Gly•Gly(SEQ ID NO: 25) Gly•Gly•Gly•Gly•Gly•Gly•Ser (SEQ ID NO: 26)Se•Gly•Gly•Gly•Gly•Gly•Gly (Gly•Gly•Gly•Gly•Ser (SEQ ID NO: 21))n(Se•Gly•Gly•Gly•Gly (SEQ ID NO: 22))nwhere n is an integer of 1 or larger. The length or sequences of peptidelinkers can be selected accordingly by those skilled in the artdepending on the purpose.

Synthetic linkers (chemical crosslinking agents) is routinely used tocrosslink peptides, and for example:

N-hydroxy succinimide (NHS),disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS³),dithiobis(succinimidyl propionate) (DSP),dithiobis(sulfosuccinimidyl propionate) (DTSSP),ethylene glycol bis(succinimidyl succinate) (EGS),ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS),disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES),and bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).These crosslinking agents are commercially available.

When multiple linkers for linking the respective domains are used, theymay all be of the same type, or may be of different types. In additionto the linkers exemplified above, linkers with peptide tags such as Histag, HA tag, myc tag, and FLAG tag may also be suitably used.Furthermore, hydrogen bonding, disulfide bonding, covalent bonding,ionic interaction, and properties of binding with each other as a resultof combination thereof may be suitably used. For example, the affinitybetween CH1 and CL of antibody may be used, and Fc regions originatingfrom the above-described bispecific antibodies may also be used forhetero Fc region association. Moreover, disulfide bonds formed betweendomains may also be suitably used.

In order to link respective domains via peptide linkage, polynucleotidesencoding the domains are linked together in frame. Known methods forlinking polynucleotides in frame include techniques such as ligation ofrestriction fragments, fusion PCR, and overlapping PCR. Such methods canbe appropriately used alone or in combination to constructantigen-binding molecules of the present invention. In the presentinvention, the terms “linked” and “fused”, or “linkage” and “fusion” areused interchangeably. These terms mean that two or more elements orcomponents such as polypeptides are linked together to form a singlestructure by any means including the above-described chemical linkingmeans and genetic recombination techniques. Fusing in frame means, whentwo or more elements or components are polypeptides, linking two or moreunits of reading frames to form a continuous longer reading frame whilemaintaining the correct reading frames of the polypeptides. When twomolecules of Fab are used as an antigen-binding domain, an antibody,which is an antigen-binding molecule of the present invention where theantigen-binding domain is linked in frame to a constant region includingan Fc region via peptide bond without linker, can be used as a preferredantigen-binding molecule of the present invention.

Low-Molecular-Weight Antibody

The antibodies used in the present invention are not limited tofull-length antibody molecules, and can be low-molecular-weightantibodies (minibodies) and modified products thereof. Alow-molecular-weight antibody includes an antibody fragment that lacks aportion of a full-length antibody (for example, whole antibody such aswhole IgG); and is not particularly limited as long as it has anantigen-binding activity. The low-molecular-weight antibody of thepresent invention is not particularly limited as long as it is a portionof a full-length antibody, but preferably comprises a heavy-chainvariable region (VH) and/or a light-chain variable region (VL). Theamino acid sequence of VH or VL may have substitution(s), deletion(s),addition(s), and/or insertion(s). Furthermore, as long as it has anantigen-binding activity, VH and/or VL can be partially deleted. Thevariable region may be chimerized or humanized. Specific examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv. Specific examplesof low-molecular-weight antibodies include Fab, Fab′, F(ab′)2, Fv, scFv(single chain Fv), diabody, and sc(Fv)2 (single chain (Fv)2). Multimersof these antibodies (for example, dimers, trimers, tetramers, andpolymers) are also included in the low-molecular-weight antibodies ofthe present invention.

Antibody fragments can be produced by treating an antibody with anenzyme such as papain and pepsin. Alternatively, genes encoding theseantibody fragments can be constructed, inserted into expression vectors,and then expressed in appropriate host cells (see, for example, Co etal., (J. Immunol. (1994) 152, 2968-2976); Better and Horwitz (Methods inEnzymology (1989) 178, 476-496), Plueckthun and Skerra (Methods inEnzymology (1989) 178, 476-496); Lamoyi (Methods in Enzymology (1989)121, 652-663); Rousseaux (Methods in Enzymology (1989) 121, 663-669);and Bird, et al., TIBTECH (1991) 9, 132-137).

A diabody refers to a bivalent low-molecular-weight antibody constructedby gene fusion (Hollinger et al., (Proc. Natl. Acad. Sci. USA 90,6444-6448 (1993)); EP 404,097; WO 1993/11161; and such). A diabody is adimer composed of two polypeptide chains. Generally, in each polypeptidechain constituting the dimer, VL and VH are linked by a linker withinthe same chain. The linker in a diabody is generally short enough toprevent binding between VL and VH. Specifically, the amino acid residuesconstituting the linker are, for example, about five residues. A linkerbetween VL and VH that are encoded by the same polypeptide chain is tooshort to form a single-chain variable region fragment, and a dimer isformed between the polypeptide chains. As a result, diabodies have twoantigen binding sites.

scFv can be obtained by linking the H-chain V region and L-chain Vregion of an antibody. In scFv, the H-chain V region and L-chain Vregion are ligated via a linker, preferably a peptide linker (Huston, etal., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The H-chain Vregion and L-chain V region of scFv may be derived from any of theantibodies described herein. The peptide linker for ligating the Vregions is not particularly limited; and for example, any single-chainpeptide consisting of 3 to 25 residues or so, or peptide linkersdescribed later or such can be used as the linker. PCR methods such asthose described above can be used for ligating the V regions. DNAencoding scFv can be amplified by a PCR method using as a templateeither whole DNA or a partial DNA encoding a desired amino acidsequence, which is selected from a DNA sequence encoding the H chain orthe H chain V region of the above-mentioned antibody, and a DNA encodingthe L chain or the L chain V region of the above-mentioned antibody; andusing a pair of primers having sequences corresponding to the sequencesof the two ends. Next, a DNA having the desired sequence can be obtainedby performing a PCR reaction using a combination of a DNA encoding thepeptide linker portion, and a pair of primers having sequences designedso that both ends of the DNA will be ligated to the H chain and the Lchain, respectively. Once the scFv-encoding DNA is constructed,expression vectors having the DNA, and recombinant cells transformedwith the expression vector can be obtained according to conventionalmethods. Furthermore, the scFvs can be obtained by culturing theresulting recombinant cells to express the scFv-encoding DNA.

sc(Fv)2 is a low-molecular-weight antibody prepared by linking two VHsand two VLs with linkers or such to form a single chain (Hudson et al.(J. Immunol. Methods 1999; 231: 177-189)). sc(Fv)2 can be produced, forexample, by linking scFvs with a linker.

Moreover, antibodies in which two VHs and two VLs are arranged in theorder of VH, VL, VH, and VL starting from the N-terminal side of asingle chain polypeptide ([VH]-linker-[VL]-linker-[VH]-linker-[VL]) arepreferred. The order of the two VHs and the two VLs is not particularlylimited to the above-mentioned arrangement, and they may be arranged inany order. Examples include the following arrangements:

-   -   [VL]-linker-[VH]-linker-[VH]-linker-[VL]    -   [VH]-linker-[VL]-linker-[VL]-linker-[VH]    -   [VH]-linker-[VH]-linker-[VL]-linker-[VL]    -   [VL]-linker-[VL]-linker-[VH]-linker-[VH]    -   [VL]-linker-[VH]-linker-[VL]-linker-[VH]

A linker similar to the linker described in the section “Antigen-bindingmolecules” above may be used as the linker for linking the antibodyvariable regions. A particularly preferred embodiment of sc(Fv)2 in thepresent invention includes, for example, the following sc(Fv)2:

-   -   [VH]-peptide linker (15 amino acids)-[VL]-peptide linker (15        amino acids)-[VH]-peptide linker (15 amino acids)-[VL]

Typically, three linkers are required to link four antibody variableregions. The linkers to be used may be of the same type or differenttypes. Examples of a non-limiting embodiment of a low-molecular-weightantibody in the present invention include a diabody or sc(Fv)2, whereinthe paratopes are different from each other; one of the paratopes bindsto an epitope in a membrane-type molecule which binds to a cancer cellmembrane; and the other paratope binds to an epitope in themembrane-type molecule expressed on the cell membrane of effector cells.In the above-mentioned diabody or sc(Fv)2, the binding activity of oneparatope that binds to an epitope in a membrane-type molecule whichbinds to a cancer cell membrane may depend on a cancer tissue-specificcompound, the binding activity of one of the paratopes toward an epitopein a membrane-type molecule which binds to an effector cell membrane maydepend on a cancer tissue-specific compound, or the binding activitiesof both paratopes may depend on a cancer tissue-specific compound.

A non-limiting embodiment of a low-molecular-weight antibody in thepresent invention includes, for example, a diabody or sc(Fv)2, whereinthe paratopes are different from each other; one of the paratopes bindsto an epitope in a membrane-type molecule which binds to a cancer cellmembrane; and the other paratope binds to an epitope in a cytotoxicsubstance. In the diabody or sc(Fv)2 mentioned above, the bindingactivity of one of the paratopes that binds to an epitope in amembrane-type molecule which binds to a cancer cell membrane may dependon a cancer tissue-specific compound, the binding activity of the otherparatope that binds to an epitope in a cytotoxic substance may depend ona cancer tissue-specific compound, or the binding activities of bothparatopes may depend on a cancer tissue-specific compound.

Such low-molecular-weight antibody can be obtained by treating anantibody with an enzyme such as papain or pepsin to generate antibodyfragments, or by constructing DNAs that encode these antibody fragmentsor low-molecular-weight antibodies, inserting them into expressionvectors, and then expressing them in appropriate host cells (see, forexample, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M.and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Pluckthun, A.and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E.,Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., MethodsEnzymol. (1986) 121, 663-669; and Bird, R. E. and Walker, B. W., TrendsBiotechnol. (1991) 9, 132-137).

FcRn

Unlike Fcγ receptor belonging to the immunoglobulin superfamily, humanFcRn is structurally similar to polypeptides of major histocompatibilitycomplex (MHC) class I, exhibiting 22% to 29% sequence identity to classI MHC molecules (Ghetie el al., Immunol. Today (1997) 18 (12): 592-598).FcRn is expressed as a heterodimer consisting of soluble β or lightchain (β2 microglobulin) complexed with transmembrane a or heavy chain.Like MHC, FcRn a chain comprises three extracellular domains (α1, α2,and α3) and its short cytoplasmic domain anchors the protein onto thecell surface. α1 and α2 domains interact with the FcRn-binding domain ofthe antibody Fc region (Raghavan et al., Immunity (1994) 1: 303-315).

FcRn is expressed in maternal placenta and york sac of mammals, and isinvolved in mother-to-fetus IgG transfer. In addition, in neonatal smallintestine of rodents, where FcRn is expressed, FcRn is involved intransfer of maternal IgG across brush border epithelium from ingestedcolostrum or milk. FcRn is expressed in a variety of other tissues andendothelial cell systems of various species. FcRn is also expressed inadult human endothelia, muscular blood vessels, and hepatic sinusoidalcapillaries. FcRn is believed to play a role in maintaining the plasmaIgG concentration by mediating recycling of IgG to serum upon binding toIgG. Typically, binding of FcRn to IgG molecules is strictly pHdependent. The optimal binding is observed in an acidic pH range below7.0.

Human FcRn whose precursor is a polypeptide having the signal sequenceof SEQ ID NO: 28 (the polypeptide with the signal sequence is shown inSEQ ID NO: 29) forms a complex with human β2-microglobulin in vivo.Soluble human FcRn complexed with β2-microglobulin is produced by usingconventional recombinant expression techniques. Fc regions of thepresent invention can be assessed for their binding activity to such asoluble human FcRn complexed with β2-microglobulin. Herein, unlessotherwise specified, human FcRn refers to a form capable of binding toan Fc region of the present invention. Examples include a complexbetween human FcRn and human β2-microglobulin.

Binding Activity of the Fc Region to FcRn, in Particular, Human FcRn

The binding activity of an Fc region of the present invention to FcRn,human FcRn in particular, can be measured by methods known to thoseskilled in the art, as described in the section “Binding Activity”above. Those skilled in the art can appropriately determine theconditions other than pH. The antigen-binding activity and humanFcRn-binding activity of an antigen-binding molecule can be assessedbased on the dissociation constant (KD), apparent dissociation constant(KD), dissociation rate (kd), apparent dissociation rate (kd), and such.These can be measured by methods known to those skilled in the art. Forexample, Biacore (GE healthcare), Scatchard plot, or flow cytometer maybe used.

When the human FcRn-binding activity of an Fc region of the presentinvention is measured, conditions other than the pH are not particularlylimited, and can be appropriately selected by those skilled in the art.Measurements can be carried out, for example, at 37° C. using MESbuffer, as described in International Publication No. WO 2009125825.Alternatively, the human FcRn-binding activity of an Fc region of thepresent invention can be measured by methods known to those skilled inthe art, and may be measured by using, for example, Biacore (GEHealthcare) or such. The binding activity of an Fc region of the presentinvention to human FcRn can be assessed by pouring, as an analyte, humanFcRn, an Fc region, or an antigen-binding molecule of the presentinvention containing the Fc region into a chip immobilized with an Fcregion, an antigen-binding molecule of the present invention containingthe Fc region, or human FcRn.

A neutral pH range as the condition where the Fc region contained in anantigen-binding molecule of the present invention has the FcRn-bindingactivity means pH6.7 to pH10.0 in general. Preferably, the neutral pHrange is a range indicated with arbitrary pH values between pH7.0 andpH8.0, and is preferably selected from pH7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, and 8.0, and is particularly preferably pH7.4 thatis close to the pH of plasma (blood) in vivo. When the binding affinitybetween the human FcRn-binding domain and human FcRn at pH7.4 is too lowto assess, pH7.0 may be used instead of pH7.4. Herein, an acidic pHrange as the condition where the Fc region contained in anantigen-binding molecule of the present invention has the FcRn-bindingactivity means pH4.0 to pH6.5 in general. Preferably, the acidic pHrange means pH5.5 to pH6.5, particularly preferably pH5.8 to pH6.0 whichis close to the pH in the early endosome in vivo. Regarding thetemperature used as the measurement condition, the binding affinitybetween the human FcRn-binding domain and human FcRn may be assessed atany temperature between 10° C. and 50° C. Preferably, the bindingaffinity between the human FcRn-binding domain and human FcRn can bedetermined at 15° C. to 40° C. More preferably, the binding affinitybetween the human FcRn-binding domain and human FcRn can be determinedin the same manner at an arbitrary temperature between 20° C. and 35°C., such as any one temperature of 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, and 35° C. In an embodiment of the presentinvention, the temperature includes, but is not limited to, for example,25° C.

According to the Journal of Immunology (2009) 182, 7663-7671, the humanFcRn-binding activity of native human IgG1 in an acidic pH range (pH6.0) is 1.7 μM (KD), and the activity is almost undetectable in aneutral pH range. Thus, in a preferred embodiment, antigen-bindingmolecules comprising an Fc region of which human FcRn-binding activityin an acidic pH range is 20 μM (KD) or stronger may be screened. In amore preferred embodiment, the antigen-binding molecules comprising anFc region of which human FcRn-binding activity in an acidic pH range is2.0 μM (KD) or stronger may be screened. In a still more preferredembodiment, the antigen-binding molecules comprising an Fc region ofwhich human FcRn-binding activity in an acidic pH range is 0.5 μM (KD)or stronger may be screened. The above-mentioned KD values aredetermined by the method described in the Journal of Immunology (2009)182: 7663-7671 (by immobilizing the antigen-binding molecule onto a chipand loading human FcRn as an analyte).

Fc Region Having FcRn-Binding Activity Under an Acidic pH RangeCondition

An Fc region having FcRn-binding activity under an acidic pH rangecondition may also be preferably used as the Fc region contained in anantigen-binding molecule provided by the present invention. Generally,IgG antibodies are known to have long plasma retention through bindingto FcRn. Binding between IgG and FcRn is observed only under acidicconditions (pH 6.0), and the binding is hardly observed under neutralconditions (pH 7.4). IgG antibodies are non-specifically incorporatedinto cells, but they return to the cell surface by binding to FcRn inthe endosome under endosomal acidic conditions, and then dissociate fromFcRn under neutral conditions in plasma. When mutations are introducedinto the Fc region of IgG to eliminate the FcRn-binding under an acidicpH range condition, the antibodies are not recycled from inside theendosome into plasma. Therefore, plasma retention of the antibody isremarkably impaired. A method for improving FcRn-binding under an acidicpH range condition has been reported as a method for improving plasmaretention of IgG antibodies. Improving FcRn-binding under an acidic pHrange condition by introducing amino acid substitutions into the IgGantibody Fc region can increase the efficiency of recycling from insidethe endosome into plasma, and as a result, plasma retention is improved.

The present invention is not restricted to a particular theory, but forexample, when an antigen-binding molecule provided by the presentinvention binds to a membrane-type antigen expressed on cancer cellscontained in cancer tissues, it may be possible to continuously suppresscancer cell proliferation as described below. Even after cancer cellsexpressing a membrane-type molecule, to which an antigen-bindingmolecule of the present invention is bound in the presence of a highconcentration of a cancer tissue-specific compound, are damaged bycytotoxic activity mediated by the antigen-binding molecule, the antigenmay still be bound to the antigen-binding domain in the antigen-bindingmolecule. From the antigen-binding molecules non-specificallyincorporated into cells, those that release the antigen in the presenceof a low concentration of the cancer tissue-specific compound return tothe cell surface by binding to FcRn in the endosome under acidicconditions inside the endosome, and then dissociate from FcRn underneutral conditions in plasma. In the presence of a high concentration ofa cancer tissue-specific compound, the antigen-binding molecules of thepresent invention recycled in this manner can bind again to theirantigens which are membrane-type molecules expressed on cancer cells.

The present invention is not restricted to a particular theory, but forexample, when a soluble antigen bound by antigen-binding moleculesprovided by the present invention is a ligand that positively regulatesactivation of inflammatory cells or proliferation of target cellscontained in a target tissue, it may be possible to suppressproliferation of target cells or activation of inflammatory cells asdescribed below. Antigen-binding molecules of the present inventionbound to the soluble molecule, i.e., its antigen, are non-specificallyincorporated into cells in the presence of a high concentration oftarget tissue-specific compounds. This is followed by release of theantigen in the presence of a low concentration of target tissue-specificcompounds, the antigen-binding molecules return to the cell surface bybinding to FcRn in the endosome under acidic conditions inside theendosome, and then the antigen-binding molecules dissociate from FcRnunder neutral conditions in plasma. The antigen-binding molecules of thepresent invention recycled in this manner can bind again to solublemolecules, i.e., their antigen, in the presence of a high concentrationof target tissue-specific compounds. On the other hand, the antigensthat dissociated from the antigen-binding molecules in the presence of alow concentration of the target tissue-specific compounds are degradedin the lysosome. The concentration of the soluble antigen decreases asit passes through the recycling stage. Therefore, it is considered thatcancer cell proliferation or inflammatory cell activation can besuppressed.

In the present invention, preferred Fc regions have an FcRn-bindingactivity in an acidic pH range condition. When an Fc region originallyhas an FcRn-binding activity under an acidic pH range condition, thedomain can be used as it is. When the domain has a weak or noFcRn-binding activity under an acidic pH range condition, an Fc regionhaving a desired FcRn-binding activity can be obtained by altering aminoacids of an antigen-binding molecule. Fc regions having a desired orenhanced FcRn-binding activity under an acidic pH range condition canalso be suitably obtained by altering the amino acids of an Fc region.Amino acid alterations of an Fc region that result in such a desiredbinding activity can be found by comparing the FcRn-binding activityunder an acidic pH range condition before and after amino acidalteration. Those skilled in the art can appropriately alter the aminoacids using known techniques similar to the aforementioned techniquesused to modify the Fcγ-receptor-binding activity.

Fc regions comprised in the antigen-binding molecules of the presentinvention, which have an FcRn-binding activity under an acidic pH rangecondition, can be obtained by any method. Specifically, FcRn-bindingdomains having an FcRn-binding activity or an enhanced FcRn-bindingactivity under an acidic pH range condition can be obtained by alteringthe amino acids of an IgG-type human immunoglobulin used as a startingFc region. Preferred Fc regions of an IgG-type immunoglobulin foralteration include, for example, those of human IgGs (IgG1, IgG2, IgG3,and IgG4, and variants thereof). As long as the Fc region has anFcRn-binding activity under an acidic pH range condition or can increasethe human FcRn-binding activity under an acidic pH range condition,amino acids at any position may be altered into other amino acids. Whenthe antigen-binding molecule contains the Fc region of human IgG1 as theFc region, it is preferable that the resulting Fc region contains analteration that results in the effect of enhancing FcRn binding under anacidic pH range condition as compared to the binding activity of thestarting human IgG1 Fc region. Amino acids that allow such alterationinclude, for example, amino acids of positions 252, 254, 256, 309, 311,315, 433, and/or 434 according to EU numbering, and their combinationamino acids at positions 253, 310, 435, and/or 426 as described in WO1997/034631. Favorable examples include amino acids of positions 238,252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311,312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400, 413,415, 424, 433, 434, 435, 436, 439, and/or 447 as indicated by EUnumbering as described in WO 2000/042072. Similarly, favorable examplesof amino acids that allow such alteration include, amino acids ofpositions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387,389, 428, 433, 434, and/or 436 according to EU numbering as described inWO 2002/060919. Furthermore, amino acids that allow such alterationinclude, for example, amino acids of positions 250, 314, and 428according to EU numbering as described in WO2004/092219. In addition,favorable examples of amino acids that allow such alteration includeamino acids of positions 238, 244, 245, 249, 252, 256, 257, 258, 260,262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 311, 312, 316, 317,318, 332, 339, 341, 343, 375, 376, 377, 378, 380, 382, 423, 427, 430,431, 434, 436, 438, 440, and/or 442 as described in WO 2006/020114.Furthermore, favorable examples of amino acids that allow suchalteration include amino acids of positions 251, 252, 307, 308, 378,428, 430, 434, and/or 436 according to EU numbering as described in WO2010/045193. Alteration of these amino acids enhances FcRn binding ofthe Fc region of an IgG-type immunoglobulin under an acidic pH rangecondition.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding under an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 includes atleast one or more amino acid alterations selected from the groupconsisting of:

Arg or Leu for the amino acid of position 251;Phe, Ser, Thr, or Tyr for the amino acid of position 252;Ser or Thr for the amino acid of position 254;Arg, Gly, Ile, or Leu for the amino acid of position 255;Ala, Arg, Asn, Asp, Gln, Glu, or Thr for the amino acid of position 256;Ile or Thr for the amino acid of position 308;Pro for the amino acid of position 309;Glu, Leu, or Ser for the amino acid of position 311;Ala or Asp for the amino acid of position 312;Ala or Leu for the amino acid of position 314;Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the amino acid of position385;Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr for the amino acid of position386;Ala, Arg, His, Pro, Ser, or Thr for the amino acid of position 387;Asn, Pro, or Ser for the amino acid of position 389;Leu, Met, Phe, Ser, or Thr for the amino acid of position 428;Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position 433;His, Phe, or Tyr for the amino acid of position 434; andArg, Asn, His, Lys, Met, or Thr for the amino acid of position 436, asindicated by EU numbering. Meanwhile, the number of amino acids to bealtered is not particularly limited; and amino acid may be altered atonly one site or amino acids may be altered at two or more sites.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding in an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 may bealterations including Ile for the amino acid of position 308, Pro forthe amino acid of position 309, and/or Glu for the amino acid ofposition 311 according to EU numbering. Another non-limiting embodimentof this alteration may include Thr for the amino acid of position 308,Pro for the amino acid of position 309, Leu for the amino acid ofposition 311, Ala for the amino acid of position 312, and/or Ala for theamino acid of position 314. Furthermore, another non-limiting embodimentof this alteration may include Ile or Thr for the amino acid of position308, Pro for the amino acid of position 309, Glu, Leu, or Ser for theamino acid of position 311, Ala for the amino acid of position 312,and/or Ala or Leu for the amino acid of position 314. Anothernon-limiting embodiment of this alteration may include Thr for the aminoacid of position 308, Pro for the amino acid of position 309, Ser forthe amino acid of position 311, Asp for the amino acid of position 312,and/or Leu for the amino acid of position 314.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding under an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 may bealterations including Leu for the amino acid of position 251, Tyr forthe amino acid of position 252, Ser or Thr for the amino acid ofposition 254, Arg for the amino acid of position 255, and/or Glu for theamino acid of position 256 according to EU numbering.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding under an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 may bealterations including Leu, Met, Phe, Ser, or Thr for the amino acid ofposition 428, Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid ofposition 433, His, Phe, or Tyr for the amino acid of position 434,and/or Arg, Asn, His, Lys, Met, or Thr for the amino acid of position436 according to EU numbering. Another non-limiting embodiment of thisalteration may include His or Met for the amino acid of position 428,and/or His or Met for the amino acid of position 434.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding under an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 may bealterations including Arg for the amino acid of position 385, Thr forthe amino acid of position 386, Arg for the amino acid of position 387,and/or Pro for the amino acid of position 389 according to EU numbering.Another non-limiting embodiment of this alteration may include Asp forthe amino acid of position 385, Pro for the amino acid of position 386,and/or Ser for the amino acid of position 389.

Furthermore, when the Fc region of human IgG1 is comprised as the Fcregion, a non-limiting embodiment of the alteration that results in theeffect of enhancing FcRn binding under an acidic pH range condition ascompared to the binding activity of the starting Fc region of human IgG1include at least one or more amino acid alterations selected from thegroup consisting of:

Gln or Glu for the amino acid of position 250; andLeu or Phe for the amino acid of position 428 according to EU numbering.The number of amino acids to be altered is not particularly limited; andamino acid may be altered at only one site or amino acids may be alteredat two sites.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding under an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 may bealterations including Gln for the amino acid of position 250, and/or Leuor Phe for the amino acid of position 428 according to EU numbering.Another non-limiting embodiment of this alteration may include Glu forthe amino acid of position 250, and/or Leu or Phe for the amino acid ofposition 428.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding under an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 include atleast two or more amino acid alterations selected from the groupconsisting of:

Asp or Glu for the amino acid of position 251;Tyr for the amino acid of position 252;Gln for the amino acid of position 307;Pro for the amino acid of position 308;Val for the amino acid of position 378;Ala for the amino acid of position 380;Leu for the amino acid of position 428;Ala or Lys for the amino acid of position 430;Ala, His, Ser, or Tyr for the amino acid of position 434; andIle for the amino acid of position 436, as indicated by EU numbering.The number of amino acids to be altered is not particularly limited; andamino acid may be altered at only two sites or amino acids may bealtered at three or more sites.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding under an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 may bealterations including Gln for the amino acid of position 307, and Ala orSer for the amino acid of position 434 according to EU numbering.Another non-limiting embodiment of this alteration may include Pro forthe amino acid of position 308, and Ala for the amino acid of position434. Furthermore, another non-limiting embodiment of this alteration mayinclude Tyr for the amino acid of position 252, and Ala for the aminoacid of position 434. A different non-limiting embodiment of thisalteration may include Val for the amino acid of position 378, and Alafor the amino acid of position 434. Another different non-limitingembodiment of this alteration may include Leu for the amino acid ofposition 428, and Ala for the amino acid of position 434. Anotherdifferent non-limiting embodiment of this alteration may include Ala forthe amino acid of position 434, and Ile for the amino acid of position436. Furthermore, another non-limiting embodiment of this alteration mayinclude Pro for the amino acid of position 308, and Tyr for the aminoacid of position 434. In addition, another non-limiting embodiment ofthis alteration may include Gln for the amino acid of position 307, andIle for the amino acid of position 436.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding under an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 may bealterations including any one of Gln for the amino acid of position 307,Ala for the amino acid of position 380, and Ser for the amino acid ofposition 434 according to EU numbering. Another non-limiting embodimentof this alteration may include Gln for the amino acid of position 307,Ala for the amino acid of position 380, and Ala for the amino acid ofposition 434. Furthermore, another non-limiting embodiment of thisalteration may include Tyr for the amino acid of position 252, Pro forthe amino acid of position 308, and Tyr for the amino acid of position434. A different non-limiting embodiment of this alteration may includeAsp for the amino acid of position 251, Gln for the amino acid ofposition 307, and His for the amino acid of position 434.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding under an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 includealteration of at least two or more amino acids selected from the groupconsisting of:

Leu for the amino acid of position 238;Leu for the amino acid of position 244;Arg for the amino acid of position 245;Pro for the amino acid of position 249;Tyr for the amino acid of position 252;Pro for the amino acid of position 256;Ala, Ile, Met, Asn, Ser, or Val for the amino acid of position 257;Asp for the amino acid of position 258;Ser for the amino acid of position 260;Leu for the amino acid of position 262;Lys for the amino acid of position 270;Leu or Arg for the amino acid of position 272;Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for theamino acid of position 279;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr,Trp, or Tyr for the amino acid of position 283;Asn for the amino acid of position 285;Phe for the amino acid of position 286;Asn or Pro for the amino acid of position 288;Val for the amino acid of position 293;Ala, Glu, or Met for the amino acid of position 307;Ala, Ile, Lys, Leu, Met, Val, or Trp for the amino acid of position 311;Pro for the amino acid of position 312;Lys for the amino acid of position 316;Pro for the amino acid of position 317;Asn or Thr for the amino acid of position 318;Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid of position332;Asn, Thr, or Trp for the amino acid of position 339;Pro for the amino acid of position 341;Glu, His, Lys, Gln, Arg, Thr, or Tyr for the amino acid of position 343;Arg for the amino acid of position 375;Gly, Ile, Met, Pro, Thr, or Val for the amino acid of position 376;Lys for the amino acid of position 377;Asp or Asn for the amino acid of position 378;Asn, Ser, or Thr for the amino acid of position 380;Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyrfor the amino acid of position 382;Asn for the amino acid of position 423;Asn for the amino acid of position 427;Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, orTyr for the amino acid of position 430;His or Asn for the amino acid of position 431;Phe, Gly, His, Trp, or Tyr for the amino acid of position 434;Ile, Leu, or Thr for the amino acid of position 436;Lys, Leu, Thr, or Trp for the amino acid of position 438;Lys for the amino acid of position 440; andLys for the amino acid of position 442 according to EU numbering. Thenumber of amino acids to be altered is not particularly limited andamino acid at only two sites may be altered and amino acids at three ormore sites may be altered.

When the Fc region of human IgG1 is comprised as the Fc region, anon-limiting embodiment of the alteration that results in the effect ofenhancing FcRn binding under an acidic pH range condition as compared tothe binding activity of the starting Fc region of human IgG1 may bealterations including Ile for the amino acid of position 257, and Ilefor the amino acid of position 311 according to EU numbering. Anothernon-limiting embodiment of this alteration may include Ile for the aminoacid of position 257, and His for the amino acid of position 434.Another non-limiting embodiment of this alteration may include Val forthe amino acid of position 376, and His for the amino acid of position434.

Fc Regions Having FcRn-Binding Activity Under Neutral pH RangeConditions

Furthermore, in another non-limiting embodiment, one may screen forantigen-binding molecules comprising an Fc region with thecharacteristic of having a human FcRn-binding activity in the neutral pHrange instead of the above-described characteristic of having a humanFcRn-binding activity in the acidic pH range. In a preferred embodiment,one may screen for antigen-binding molecules comprising an Fc regionwhose human FcRn-binding activity in the neutral pH range is 40 μM (KD)or stronger. In a more preferred embodiment, one may screen forantigen-binding molecules comprising an Fc region whose humanFcRn-binding activity in the neutral pH range is 15 μM (KD) or stronger.

Furthermore, in another non-limiting embodiment, one may screen forantigen-binding molecules comprising an Fc region with thecharacteristic of having a human FcRn-binding activity in the neutral pHrange in addition to the above-described characteristic of having ahuman FcRn-binding activity in the acidic pH range. In a preferredembodiment, one may screen for antigen-binding molecules comprising anFc region whose human FcRn-binding activity in the neutral pH range is40 μM (KD) or stronger. In a more preferred embodiment, one may screenfor antigen-binding molecules comprising an Fc region whose humanFcRn-binding activity in the neutral pH range is 15 μM (KD) or stronger.

In the present invention, preferred Fc regions have a human FcRn-bindingactivity in the acidic pH range and/or neutral pH range. When an Fcregion originally has a human FcRn-binding activity in the acidic pHrange and/or neutral pH range, it can be used as it is. When an Fcregion has a weak or no human FcRn-binding activity in the acidic pHrange and/or neutral pH range, antigen-binding molecules comprising anFc region having a desired human FcRn-binding activity can be obtainedby altering amino acids of the Fc region comprised in theantigen-binding molecules. Fc regions having a desired humanFcRn-binding activity in the acidic pH range and/or neutral pH range canalso be suitably obtained by altering amino acids of a human Fc region.Alternatively, antigen-binding molecules comprising an Fc region havinga desired human FcRn-binding activity can be obtained by altering aminoacids of an Fc region that originally has a human FcRn-binding activityin the acidic pH range and/or neutral pH range. Amino acid alterationsof a human Fc region that result in such a desired binding activity canbe found by comparing the human FcRn-binding activity in the acidic pHrange and/or neutral pH range before and after amino acid alteration.Those skilled in the art can appropriately alter amino acids using knownmethods.

In the present invention, “alteration of amino acids” or “amino acidalteration” of an Fc region includes alteration into an amino acidsequence which is different from that of the starting Fc region. Thestarting Fc region may be any Fc region, as long as a variant modifiedfrom the starting Fc region can bind to human FcRn in an acidic pH range(i.e., the starting Fc region does not necessarily need to have anactivity to bind to human FcRn in a neutral pH range). Examples ofstarting Fc regions preferably include Fc regions of IgG antibodies,i.e., native Fc regions. Furthermore, an altered Fc region modified froma starting Fc region which has been already modified can also be usedpreferably as an altered Fc region of the present invention. The“starting Fc region” can refer to the polypeptide itself, a compositioncomprising the starting Fc region, or an amino acid sequence encodingthe starting Fc region. Starting Fc regions can comprise a known IgGantibody Fc region produced via recombination described briefly insection “Antibodies”. The origin of starting Fc regions is not limited,and they may be obtained from human or any nonhuman organisms. Suchorganisms preferably include mice, rats, guinea pigs, hamsters, gerbils,cats, rabbits, dogs, goats, sheep, bovines, horses, camels and organismsselected from nonhuman primates. In another embodiment, starting Fcregions can also be obtained from cynomolgus monkeys, marmosets, rhesusmonkeys, chimpanzees, or humans. Starting Fc regions can be obtainedpreferably from human IgG1; however, they are not limited to anyparticular IgG subclass. This means that an Fc region represented byhuman IgG1 (SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), orIgG4 (SEQ ID NO: 8) can be used appropriately as a starting Fc region,and herein also means that an Fc region of an arbitrary IgG class orsubclass derived from any organisms described above can be preferablyused as a starting Fc region. Examples of naturally-occurring IgGvariants or altered forms are described in published documents (Curr.Opin. Biotechnol. (2009) 20 (6): 685-91; Curr. Opin. Immunol. (2008) 20(4), 460-470; Protein Eng. Des. Sel. (2010) 23 (4): 195-202;International Publication Nos. WO 2009/086320, WO 2008/092117, WO2007/041635, and WO 2006/105338); however, they are not limited to theexamples.

Examples of alterations include those with one or more mutations, forexample, mutations by substitution of different amino acid residues foramino acids of starting Fc regions, by insertion of one or more aminoacid residues into amino acids of starting Fc regions, or by deletion ofone or more amino acids from amino acids of starting Fc regions.Preferably, the amino acid sequences of altered Fc regions comprise atleast a part of the amino acid sequence of a non-native Fc region. Suchvariants must have sequence identity or similarity of less than 100% totheir starting Fc region. In a preferred embodiment, the variants haveamino acid sequence identity or similarity of about 75% to less than100%, more preferably about 80% to less than 100%, even more preferablyabout 85% to less than 100%, still more preferably about 90% to lessthan 100%, and yet more preferably about 95% to less than 100% to theamino acid sequence of their starting Fc region. In a non-limitingembodiment of the present invention, at least one amino acid isdifferent between an altered Fc region of the present invention and itsstarting Fc region. Amino acid difference between an altered Fc regionand its starting Fc region can also be preferably specified based onamino acid differences at the above-described particular amino acidresidue positions as indicated by EU numbering. Methods for producingsuch variants are exemplified in the section “Amino acid alterations”.

Fc regions comprised in the antigen-binding molecules of the presentinvention that have a human FcRn-binding activity in the neutral pHrange can be obtained by any method. Specifically, one can screen forantigen-binding molecules comprising an Fc region of which humanFcRn-binding activity in the neutral pH range is 20 μM (KD) or stronger;in a more favorable embodiment, an Fc region of which human FcRn-bindingactivity in the neutral pH range is 2.0 μM (KD) or stronger; and in aneven more favorable embodiment, an Fc region of which human FcRn-bindingactivity in the neutral pH range is 0.5 μM (KD) or stronger as a resultof altering amino acids of an IgG-type human immunoglobulin used as astarting Fc region. Preferred Fc regions of IgG-type immunoglobulins foralteration include, for example, those of human IgGs such as IgG1, IgG2,IgG3, and IgG4 shown in SEQ ID NOs: 5, 6, 7, and 8, respectively, andvariants thereof.

When an antigen-binding molecule comprises the Fc region of human IgG1as the Fc region, suitable examples of amino acids that may be alteredto achieve the above-mentioned desired effects on FcRn binding under aneutral pH range condition by altering amino acids of an IgG-type humanimmunoglobulin as a starting Fc region, include amino acids of positions238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386, 388, 400,413, 415, 424, 433, 434, 435, 436, 439, and/or 447 according to EUnumbering as described in WO 2000/042072. Similarly, favorable examplesof amino acids that allow such alteration include amino acids ofpositions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387,389, 428, 433, 434, and/or 436 according to EU numbering as described inWO 2002/060919. Furthermore, amino acids that allow such alterationinclude, for example, amino acids of positions 250, 314, and 428according to EU numbering as described in WO2004/092219. Furthermore,favorable examples of amino acids that allow such alteration includeamino acids of positions 251, 252, 307, 308, 378, 428, 430, 434, and/or436 according to EU numbering as described in WO 2010/045193. Alterationof these amino acids enhances FcRn binding of the Fc region of anIgG-type immunoglobulin under a neutral pH range condition.

Fc regions having human FcRn-binding activity in the neutral pH rangecan also be obtained by altering amino acids of human immunoglobulin ofIgG type used as the starting Fc region. The Fc regions of IgG typeimmunoglobulins adequate for alteration include, for example, those ofhuman IgGs such as IgG1, IgG2, IgG3, and IgG4 respectively representedby SEQ ID NOs: 5, 6, 7, and 8, and altered forms thereof. Amino acids ofany positions may be altered into other amino acids, as long as the Fcregions have the human FcRn-binding activity in the neutral pH range orcan increase the human FcRn-binding activity in the neutral range. Whenthe antigen-binding molecule contains the Fc region of human IgG1 as thehuman Fc region, it is preferable that the resulting Fc region containsa alteration that results in the effect of enhancing the human FcRnbinding in the neutral pH range as compared to the binding activity ofthe starting Fc region of human IgG1. Amino acids that allow suchalteration include, for example, amino acids of the following positions:221 to 225, 227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262to 272, 274, 276, 278 to 289, 291 to 312, 315 to 320, 324, 325, 327 to339, 341, 343, 345, 360, 362, 370, 375 to 378, 380, 382, 385 to 387,389, 396, 414, 416, 423, 424, 426 to 438, 440, and 442 according to EUnumbering. Alteration of these amino acids augments the human FcRnbinding of the Fc region of IgG-type immunoglobulin in the neutral pHrange.

From those described above, alterations that augment the human FcRnbinding in the neutral pH range are appropriately selected for use inthe present invention. Particularly preferred amino acids of the alteredFc regions include, for example, amino acids of positions 237, 248, 250,252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307,308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384,385, 386, 387, 389, 424, 428, 433, 434, and 436 according to the EUnumbering system. The human FcRn-binding activity in the neutral pHrange of the Fc region contained in an antigen-binding molecule can beincreased by substituting at least one amino acid selected from theabove amino acids into a different amino acid.

Particularly preferred alterations include, for example:

Met for the amino acid at position 237;Ile for the amino acid at position 248;Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr for the amino acid atposition 250;Phe, Trp, or Tyr for the amino acid at position 252;Thr for the amino acid at position 254;Glu for the amino acid at position 255;Asp, Asn, Glu, or Gln for the amino acid at position 256;Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val for the amino acid atposition 257;His for the amino acid at position 258:Ala for the amino acid at position 265;Ala or Glu for the amino acid at position 286;His for the amino acid at position 289;Ala for the amino acid at position 297;Ala for the amino acid at position 303;Ala for the amino acid at position 305;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Val, Trp, or Tyr for the amino acid at position 307;Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr for the amino acid at position308; Ala, Asp, Glu, Pro, or Arg for the amino acid at position 309;Ala, His, or Ile for the amino acid at position 311;Ala or His for the amino acid at position 312;Lys or Arg for the amino acid at position 314;Ala, Asp, or His for the amino acid at position 315;Ala for the amino acid at position 317;Val for the amino acid at position 332;Leu for the amino acid at position 334;His for the amino acid at position 360;Ala for the amino acid at position 376;Ala for the amino acid at position 380;Ala for the amino acid at position 382;Ala for the amino acid at position 384;Asp or His for the amino acid at position 385;Pro for the amino acid at position 386;Glu for the amino acid at position 387;Ala or Ser for the amino acid at position 389;Ala for the amino acid at position 424;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 428;Lys for the amino acid at position 433;Ala, Phe, His, Ser, Trp, or Tyr for the amino acid at position 434; andHis, Ile, Leu, Phe, Thr, or Val for the amino acid at position 436 ofthe Fc region according to EU numbering. Meanwhile, the number of aminoacids to be altered is not particularly limited and an amino acid atonly one site may be altered, and amino acids at two or more sites maybe altered. These combinations of amino acid alterations include, forexample, those described in Tables 2-1 to 2-33.

TABLE 2-1 Variant KD (M) Amino acid alteration site F1 8.10E−07 N434W F23.20E−06 M252Y/S254T/T256E F3 2.50E−06 N434Y F4 5.80E−06 N434S F56.80E−06 N434A F7 5.60E−06 M252Y F8 4.20E−06 M252W F9 1.40E−07M252Y/S254T/T256E/N434Y F10 6.90E−08 M252Y/S254T/T256E/N434W F113.10E−07 M252Y/N434Y F12 1.70E−07 M252Y/N434W F13 3.20E−07 M252W/N434YF14 1.80E−07 M252W/N434W F19 4.60E−07 P257L/N434Y F20 4.60E−07V308F/N434Y F21 3.00E−08 M252Y/V308P/N434Y F22 2.00E−06 M428L/N434S F259.20E−09 M252Y/S254T/T256E/V308P/N434W F26 1.00E−06 I322V F27 7.40E−06G237M F29 1.40E−06 I332V/N434Y F31 2.80E−06 G237M/V308F F32 8.00E−07S254T/N434W F33 2.30E−06 S254T/N434Y F34 2.80E−07 T256E/N434W F358.40E−07 T256E/N434Y F36 3.60E−07 S254T/T256E/N434W F37 1.10E−06S254T/T256E/N434Y F38 1.00E−07 M252Y/S254T/N434W F39 3.00E−07M252Y/S254T/N434Y F40 8.20E−08 M252Y/T256E/N434W F41 1.50E−07M252Y/T256E/N434YTable 2-2 is a continuation of Table 2-1.

TABLE 2-2 F42 1.00E−06 M252Y/S254T/T256E/N434A F43 1.70E−06 M252Y/N434AF44 1.10E−06 M252W/N434A F47 2.40E−07 M252Y/T256Q/N434W F48 3.20E−07M252Y/T256Q/N434Y F49 5.10E−07 M252F/T256D/N434W F50 1.20E−06M252F/T256D/N434Y F51 8.10E−06 N434F/Y436H F52 3.10E−06H433K/N434F/Y436H F53 1.00E−06 I332V/N434W F54 8.40E−08 V308P/N434W F569.40E−07 I332V/M428L/N434Y F57 1.10E−05 G385D/Q386P/N389S F58 7.70E−07G385D/Q386P/N389S/N434W F59 2.40E−06 G385D/Q386P/N389S/N434Y F601.10E−05 G385H F61 9.70E−07 G385H/N434W F62 1.90E−06 G385H/N434Y F632.50E−06 N434F F64 5.30E−06 N434H F65 2.90E−07 M252Y/S254T/T256E/N434FF66 4.30E−07 M252Y/S254T/T256E/N434H F67 6.30E−07 M252Y/N434F F689.30E−07 M252Y/N434H F69 5.10E−07 M428L/N434W F70 1.50E−06 M428L/N434YF71 8.30E−08 M252Y/S254T/T256E/M428L/N434W F72 2.00E−07M252Y/S254T/T256E/M428L/N434Y F73 1.70E−07 M252Y/M428L/N434W F744.60E−07 M252Y/M428L/N434Y F75 1.40E−06 M252Y/M428L/N434A F76 1.00E−06M252Y/S254T/T256E/M428L/N434A F77 9.90E−07 T256E/M428L/N434Y F787.80E−07 S254T/M428L/N434WTable 2-3 is a continuation of Table 2-2.

TABLE 2-3 F79 5.90E−06 S254T/T256E/N434A F80 2.70E−06 M252Y/T256Q/N434AF81 1.60E−06 M252Y/T256E/N434A F82 1.10E−06 T256Q/N434W F83 2.60E−06T256Q/N434Y F84 2.80E−07 M252W/T256Q/N434W F85 5.50E−07M252W/T256Q/N434Y F86 1.50E−06 S254T/T256Q/N434W F87 4.30E−06S254T/T256Q/N434Y F88 1.90E−07 M252Y/S254T/T256Q/N434W F89 3.60E−07M252Y/S254T/T256Q/N434Y F90 1.90E−08 M252Y/T256E/V308P/N434W F914.80E−08 M252Y/V308P/M428L/N434Y F92 1.10E−08M252Y/S254T/T256E/V308P/M428L/N434W F93 7.40E−07 M252W/M428L/N434W F943.70E−07 P257L/M428L/N434Y F95 2.60E−07 M252Y/S254T/T256E/M428L/N434FF99 6.20E−07 M252Y/T256E/N434H F101 1.10E−07 M252W/T256Q/P257L/N434YF103 4.40E−08 P238A/M252Y/V308P/N434Y F104 3.70E−08M252Y/D265A/V308P/N434Y F105 7.50E−08 M252Y/T307A/V308P/N434Y F1063.70E−08 M252Y/V303A/V308P/N434Y F107 3.40E−08 M252Y/V308P/D376A/N434YF108 4.10E−08 M252Y/V305A/V308P/N434Y F109 3.20E−08M252Y/V308P/Q311A/N434Y F111 3.20E−08 M252Y/V308P/K317A/N434Y F1126.40E−08 M252Y/V308P/E380A/N434Y F113 3.20E−08 M252Y/V308P/E382A/N434YF114 3.80E−08 M252Y/V308P/S424A/N434Y F115 6.60E−06 T307A/N434A F1168.70E−06 E380A/N434A F118 1.40E−05 M428L F119 5.40E−06 T250Q/M428LTable 2-4 is a continuation of Table 2-3.

TABLE 2-4 F120 6.30E−08 P257L/V308P/M428L/N434Y F121 1.50E−08M252Y/T256E/V308P/M428L/N434W F122 1.20E−07 M252Y/T256E/M428L/N434W F1233.00E−08 M252Y/T256E/V308P/N434Y F124 2.90E−07 M252Y/T256E/M428L/N434YF125 2.40E−08 M252Y/S254T/T256E/V308P/M428L/N434Y F128 1.70E−07P257L/M428L/N434W F129 2.20E−07 P257A/M428L/N434Y F131 3.00E−06P257G/M428L/N434Y F132 2.10E−07 P257I/M428L/N434Y F133 4.10E−07P257M/M428L/N434Y F134 2.70E−07 P257N/M428L/N434Y F135 7.50E−07P257S/M428L/N434Y F136 3.80E−07 P257T/M428L/N434Y F137 4.60E−07P257V/M428L/N434Y F139 1.50E−08 M252W/V308P/N434W F140 3.60E−08S239K/M252Y/V308P/N434Y F141 3.50E−08 M252Y/S298G/V308P/N434Y F1423.70E−08 M252Y/D270F/V308P/N434Y F143 2.00E−07 M252Y/V308A/N434Y F1455.30E−08 M252Y/V308F/N434Y F147 2.40E−07 M252Y/V308I/N434Y F149 1.90E−07M252Y/V308L/N434Y F150 2.00E−07 M252Y/V308M/N434Y F152 2.70E−07M252Y/V308Q/N434Y F154 1.80E−07 M252Y/V308T/N434Y F157 1.50E−07P257A/V308P/M428L/N434Y F158 5.90E−08 P257T/V308P/M428L/N434Y F1594.40E−08 P257V/V308P/M428L/N434Y F160 8.50E−07 M252W/M428I/N434Y F1621.60E−07 M252W/M428Y/N434Y F163 4.20E−07 M252W/M428F/N434Y F164 3.70E−07P238A/M252W/N434Y F165 2.90E−07 M252W/D265A/N434YTable 2-5 is a continuation of Table 2-4.

TABLE 2-5 F166 1.50E−07 M252W/T307Q/N434Y F167 2.90E−07M252W/V303A/N434Y F168 3.20E−07 M252W/D376A/N434Y F169 2.90E−07M252W/V305A/N434Y F170 1.70E−07 M252W/Q311A/N434Y F171 1.90E−07M252W/D312A/N434Y F172 2.20E−07 M252W/K317A/N434Y F173 7.70E−07M252W/E380A/N434Y F174 3.40E−07 M252W/E382A/N434Y F175 2.70E−07M252W/S424A/N434Y F176 2.90E−07 S239K/M252W/N434Y F177 2.80E−07M252W/S298G/N434Y F178 2.70E−07 M252W/D270F/N434Y F179 3.10E−07M252W/N325G/N434Y F182 6.60E−08 P257A/M428L/N434W F183 2.20E−07P257T/M428L/N434W F184 2.70E−07 P257V/M428L/N434W F185 2.60E−07M252W/I332V/N434Y F188 3.00E−06 P257I/Q311I F189 1.90E−07M252Y/T307A/N434Y F190 1.10E−07 M252Y/T307Q/N434Y F191 1.60E−07P257L/T307A/M428L/N434Y F192 1.10E−07 P257A/T307A/M428L/N434Y F1938.50E−08 P257T/T307A/M428L/N434Y F194 1.20E−07 P257V/T307A/M428L/N434YF195 5.60E−08 P257L/T307Q/M428L/N434Y F196 3.50E−08P257A/T307Q/M428L/N434Y F197 3.30E−08 P257T/T307Q/M428L/N434Y F1984.80E−08 P257V/T307Q/M428L/N434Y F201 2.10E−07 M252Y/T307D/N434Y F2032.40E−07 M252Y/T307F/N434Y F204 2.10E−07 M252Y/T307G/N434Y F205 2.00E−07M252Y/T307H/N434Y F206 2.30E−07 M252Y/T307I/N434YTable 2-6 is a continuation of Table 2-5.

TABLE 2-6 F207 9.40E−07 M252Y/T307K/N434Y F208 3.90E−07M252Y/T307L/N434Y F209 1.30E−07 M252Y/T307M/N434Y F210 2.90E−07M252Y/T307N/N434Y F211 2.40E−07 M252Y/T307P/N434Y F212 6.80E−07M252Y/T307R/N434Y F213 2.30E−07 M252Y/T307S/N434Y F214 1.70E−07M252Y/T307V/N434Y F215 9.60E−08 M252Y/T307W/N434Y F216 2.30E−07M252Y/T307Y/N434Y F217 2.30E−07 M252Y/K334L/N434Y F218 2.60E−07M252Y/G385H/N434Y F219 2.50E−07 M252Y/T289H/N434Y F220 2.50E−07M252Y/Q311H/N434Y F221 3.10E−07 M252Y/D312H/N434Y F222 3.40E−07M252Y/N315H/N434Y F223 2.70E−07 M252Y/K360H/N434Y F225 1.50E−06M252Y/L314R/N434Y F226 5.40E−07 M252Y/L314K/N434Y F227 1.20E−07M252Y/N286E/N434Y F228 2.30E−07 M252Y/L309E/N434Y F229 5.10E−07M252Y/R255E/N434Y F230 2.50E−07 M252Y/P387E/N434Y F236 8.90E−07K248I/M428L/N434Y F237 2.30E−07 M252Y/M428A/N434Y F238 7.40E−07M252Y/M428D/N434Y F240 7.20E−07 M252Y/M428F/N434Y F241 1.50E−06M252Y/M428G/N434Y F242 8.50E−07 M252Y/M428H/N434Y F243 1.80E−07M252Y/M428I/N434Y F244 1.30E−06 M252Y/M428K/N434Y F245 4.70E−07M252Y/M428N/N434Y F246 1.10E−06 M252Y/M428P/N434Y F247 4.40E−07M252Y/M428Q/N434YTable 2-7 is a continuation of Table 2-6.

TABLE 2-7 F249 6.40E−07 M252Y/M428S/N434Y F250 2.90E−07M252Y/M428T/N434Y F251 1.90E−07 M252Y/M428V/N434Y F252 1.00E−06M252Y/M428W/N434Y F253 7.10E−07 M252Y/M428Y/N434Y F254 7.50E−08M252W/T307Q/M428Y/N434Y F255 1.10E−07 M252W/Q311A/M428Y/N434Y F2565.40E−08 M252W/T307Q/Q311A/M428Y/N434Y F257 5.00E−07M252Y/T307A/M428Y/N434Y F258 3.20E−07 M252Y/T307Q/M428Y/N434Y F2592.80E−07 M252Y/D270F/N434Y F260 1.30E−07 M252Y/T307A/Q311A/N434Y F2618.40E−08 M252Y/T307Q/Q311A/N434Y F262 1.90E−07 M252Y/T307A/Q311H/N434YF263 1.10E−07 M252Y/T307Q/Q311H/N434Y F264 2.80E−07 M252Y/E382A/N434YF265 6.80E−07 M252Y/E382A/M428Y/N434Y F266 4.70E−07M252Y/T307A/E382A/M428Y/N434Y F267 3.20E−07M252Y/T307Q/E382A/M428Y/N434Y F268 6.30E−07 P238A/M252Y/M428F/N434Y F2695.20E−07 M252Y/V305A/M428F/N434Y F270 6.60E−07 M252Y/N325G/M428F/N434YF271 6.90E−07 M252Y/D376A/M428F/N434Y F272 6.80E−07M252Y/E380A/M428F/N434Y F273 6.50E−07 M252Y/E382A/M428F/N434Y F2747.60E−07 M252Y/E380A/E382A/M428F/N434Y F275 4.20E−08S239K/M252Y/V308P/E382A/N434Y F276 4.10E−08M252Y/D270F/V308P/E382A/N434Y F277 1.30E−07S239K/M252Y/V308P/M428Y/N434Y F278 3.00E−08M252Y/T307Q/V308P/E382A/N434Y F279 6.10E−08M252Y/V308P/Q311H/E382A/N434Y F280 4.10E−08S239K/M252Y/D270F/V308P/N434Y F281 9.20E−08M252Y/V308P/E382A/M428F/N434Y F282 2.90E−08M252Y/V308P/E382A/M428L/N434YTable 2-8 is a continuation of Table 2-7.

TABLE 2-8 F283 1.00E−07 M252Y/V308P/E382A/M428Y/N434Y F284 1.00E−07M252Y/V308P/M428Y/N434Y F285 9.90E−08 M252Y/V308P/M428F/N434Y F2861.20E−07 S239K/M252Y/V308P/E382A/M428Y/N434Y F287 1.00E−07M252Y/V308P/E380A/E382A/M428F/N434Y F288 1.90E−07M252Y/T256E/E382A/N434Y F289 4.80E−07 M252Y/T256E/M428Y/N434Y F2904.60E−07 M252Y/T256E/E382A/M428Y/N434Y F292 2.30E−08S239K/M252Y/V308P/E382A/M428I/N434Y F293 5.30E−08M252Y/V308P/E380A/E382A/M428I/N434Y F294 1.10E−07S239K/M252Y/V308P/M428F/N434Y F295 6.80E−07S239K/M252Y/E380A/E382A/M428F/N434Y F296 4.90E−07M252Y/Q311A/M428Y/N434Y F297 5.10E−07 M252Y/D312A/M428Y/N434Y F2984.80E−07 M252Y/Q311A/D312A/M428Y/N434Y F299 9.40E−08S239K/M252Y/V308P/Q311A/M428Y/N434Y F300 8.30E−08S239K/M252Y/V308P/D312A/M428Y/N434Y F301 7.20E−08S239K/M252Y/V308P/Q311A/D312A/M428Y/N434Y F302 1.90E−07M252Y/T256E/T307P/N434Y F303 6.70E−07 M252Y/T307P/M428Y/N434Y F3041.60E−08 M252W/V308P/M428Y/N434Y F305 2.70E−08M252Y/T256E/V308P/E382A/N434Y F306 3.60E−08 M252W/V308P/E382A/N434Y F3073.60E−08 S239K/M252W/V308P/E382A/N434Y F308 1.90E−08S239K/M252W/V308P/E382A/M428Y/N434Y F310 9.40E−08S239K/M252W/V308P/E382A/M428I/N434Y F311 2.80E−08S239K/M252W/V308P/M428F/N434Y F312 4.50E−07S239K/M252W/E380A/E382A/M428F/N434Y F313 6.50E−07S239K/M252Y/T307P/M428Y/N434Y F314 3.20E−07M252Y/T256E/Q311A/D312A/M428Y/N434Y F315 6.80E−07S239K/M252Y/M428Y/N434Y F316 7.00E−07 S239K/M252Y/D270F/M428Y/N434Y F3171.10E−07 S239K/M252Y/D270F/V308P/M428Y/N434Y F318 1.80E−08S239K/M252Y/V308P/M428I/N434YTable 2-9 is a continuation of Table 2-8.

TABLE 2-9 F320 2.00E−08 S239K/M252Y/V308P/N325G/E382A/M428I/N434Y F3213.20E−08 S239K/M252Y/D270F/V308P/N325G/N434Y F322 9.20E−08S239K/M252Y/D270F/T307P/V308P/N434Y F323 2.70E−08S239K/M252Y/T256E/D270F/V308P/N434Y F324 2.80E−08S239K/M252Y/D270F/T307Q/V308P/N434Y F325 2.10E−08S239K/M252Y/D270F/T307Q/V308P/Q311A/N434Y F326 7.50E−08S239K/M252Y/D270F/T307Q/Q311A/N434Y F327 6.50E−08S239K/M252Y/T256E/D270F/T307Q/Q311A/N434Y F328 1.90E−08S239K/M252Y/D270F/V308P/M428I/N434Y F329 1.20E−08S239K/M252Y/D270F/N286E/V308P/N434Y F330 3.60E−08S239K/M252Y/D270F/V308P/L309E/N434Y F331 3.00E−08S239K/M252Y/D270F/V308P/P387E/N434Y F333 7.40E−08S239K/M252Y/D270F/T307Q/L309E/Q311A/N434Y F334 1.90E−08S239K/M252Y/D270F/V308P/N325G/M428I/N434Y F335 1.50E−08S239K/M252Y/T256E/D270F/V308P/M428I/N434Y F336 1.40E−08S239K/M252Y/D270F/T307Q/V308P/Q311A/M428I/ N434Y F337 5.60E−08S239K/M252Y/D270F/T307Q/Q311A/M428I/N434Y F338 7.70E−09S239K/M252Y/D270F/N286E/V308P/M428I/N434Y F339 1.90E−08S239K/M252Y/D270F/V308P/L309E/M428I/N434Y F343 3.20E−08S239K/M252Y/D270F/V308P/M428L/N434Y F344 3.00E−08S239K/M252Y/V308P/M428L/N434Y F349 1.50E−07S239K/M252Y/V308P/L309P/M428L/N434Y F350 1.70E−07S239K/M252Y/V308P/L309R/M428L/N434Y F352 6.00E−07S239K/M252Y/L309P/M428L/N434Y F353 1.10E−06S239K/M252Y/L309R/M428L/N434Y F354 2.80E−08S239K/M252Y/T307Q/V308P/M428L/N434Y F356 3.40E−08S239K/M252Y/D270F/V308P/L309E/P387E/N434Y F357 1.60E−08S239K/M252Y/T256E/D270F/V308P/N325G/M428I/ N434Y F358 1.00E−07S239K/M252Y/T307Q/N434Y F359 4.20E−07 P257V/T307Q/M428I/N434Y F3601.30E−06 P257V/T307Q/M428V/N434Y F362 5.40E−08P257V/T307Q/N325G/M428L/N434Y F363 4.10E−08P257V/T307Q/Q311A/M428L/N434Y F364 3.50E−08P257V/T307Q/Q311A/N325G/M428L/N434YTable 2-10 is a continuation of Table 2-9.

TABLE 2-10 F365 5.10E−08 P257V/V305A/T307Q/M428L/N434Y F367 1.50E−08S239K/M252Y/E258H/D270F/T307Q/V308P/Q311A/ N434Y F368 2.00E−08S239K/M252Y/D270F/V308P/N325G/E382A/M428I/ N434Y F369 7.50E−08M252Y/P257V/T307Q/M428I/N434Y F372 1.30E−08S239K/M252W/V308P/M428Y/N434Y F373 1.10E−08S239K/M252W/V308P/Q311A/M428Y/N434Y F374 1.20E−08S239K/M252W/T256E/V308P/M428Y/N434Y F375 5.50E−09S239K/M252W/N286E/V308P/M428Y/N434Y F376 9.60E−09S239K/M252Y/T256E/D270F/N286E/V308P/N434Y F377 1.30E−07S239K/M252W/T307P/M428Y/N434Y F379 9.00E−09S239K/M252W/T256E/V308P/Q311A/M428Y/N434Y F380 5.60E−09S239K/M252W/T256E/N286E/V308P/M428Y/N434Y F381 1.10E−07P257V/T307A/Q311A/M428L/N434Y F382 8.70E−08P257V/V305A/T307A/M428L/N434Y F386 3.20E−08 M252Y/V308P/L309E/N434Y F3871.50E−07 M252Y/V308P/L309D/N434Y F388 7.00E−08 M252Y/V308P/L309A/N434YF389 1.70E−08 M252W/V308P/L309E/M428Y/N434Y F390 6.80E−08M252W/V308P/L309D/M428Y/N434Y F391 3.60E−08M252W/V308P/L309A/M428Y/N434Y F392 6.90E−09S239K/M252Y/N286E/V308P/M428I/N434Y F393 1.20E−08S239K/M252Y/N286E/V308P/N434Y F394 5.30E−08S239K/M252Y/T307Q/Q311A/M428I/N434Y F395 2.40E−08S239K/M252Y/T256E/V308P/N434Y F396 2.00E−08S239K/M252Y/D270F/N286E/T307Q/Q311A/M428I/ N434Y F397 4.50E−08S239K/M252Y/D270F/T307Q/Q311A/P387E/M428I/ N434Y F398 4.40E−09S239K/M252Y/D270F/N286E/T307Q/V308P/Q311A/ M428I/N434Y F399 6.50E−09S239K/M252Y/D270F/N286E/T307Q/V308P/M428I/ N434Y F400 6.10E−09S239K/M252Y/D270F/N286E/V308P/Q311A/M428I/ N434Y F401 6.90E−09S239K/M252Y/D270F/N286E/V308P/P387E/M428I/ N434Y F402 2.30E−08P257V/T307Q/M428L/N434W F403 5.10E−08 P257V/T307A/M428L/N434W F4049.40E−08 P257A/T307Q/L309P/M428L/N434Y F405 1.70E−07P257V/T307Q/L309P/M428L/N434YTable 2-11 is a continuation of Table 2-10.

TABLE 2-11 F406 1.50E−07 P257A/T307Q/L309R/M428L/N434Y F407 1.60E−07P257V/T307Q/L309R/M428L/N434Y F408 2.50E−07 P257V/N286E/M428L/N434Y F4092.00E−07 P257V/P387E/M428L/N434Y F410 2.20E−07 P257V/T307H/M428L/N434YF411 1.30E−07 P257V/T307N/M428L/N434Y F412 8.80E−08P257V/T307G/M428L/N434Y F413 1.20E−07 P257V/T307P/M428L/N434Y F4141.10E−07 P257V/T307S/M428L/N434Y F415 5.60E−08P257V/N286E/T307A/M428L/N434Y F416 9.40E−08P257V/T307A/P387E/M428L/N434Y F418 6.20E−07S239K/M252Y/T307P/N325G/M428Y/N434Y F419 1.60E−07M252Y/T307A/Q311H/K360H/N434Y F420 1.50E−07M252Y/T307A/Q311H/P387E/N434Y F421 1.30E−07M252Y/T307A/Q311H/M428A/N434Y F422 1.80E−07M252Y/T307A/Q311H/E382A/N434Y F423 8.40E−08 M252Y/T307W/Q311H/N434Y F4249.40E−08 S239K/P257A/V308P/M428L/N434Y F425 8.00E−08P257A/V308P/L309E/M428L/N434Y F426 8.40E−08 P257V/T307Q/N434Y F4271.10E−07 M252Y/P257V/T307Q/M428V/N434Y F428 8.00E−08M252Y/P257V/T307Q/M428L/N434Y F429 3.70E−08 M252Y/P257V/T307Q/N434Y F4308.10E−08 M252Y/P257V/T307Q/M428Y/N434Y F431 6.50E−08M252Y/P257V/T307Q/M428F/N434Y F432 9.20E−07P257V/T307Q/Q311A/N325G/M428V/N434Y F433 6.00E−08P257V/T307Q/Q311A/N325G/N434Y F434 2.00E−08P257V/T307Q/Q311A/N325G/M428Y/N434Y F435 2.50E−08P257V/T307Q/Q311A/N325G/M428F/N434Y F436 2.50E−07P257A/T307Q/M428V/N434Y F437 5.70E−08 P257A/T307Q/N434Y F438 3.60E−08P257A/T307Q/M428Y/N434Y F439 4.00E−08 P257A/T307Q/M428F/N434Y F4401.50E−08 P257V/N286E/T307Q/Q311A/N325G/M428L/N434YTable 2-12 is a continuation of Table 2-11.

TABLE 2-12 F441 1.80E−07 P237A/Q311A/M428L/N434Y F442 2.00E−07P257A/Q311H/M428L/N434Y F443 5.50E−08 P257A/T307Q/Q311A/M428L/N434Y F4441.40E−07 P257A/T307A/Q311A/M428L/N434Y F445 6.20E−08P257A/T307Q/Q311H/M428L/N434Y F446 1.10E−07P257A/T307A/Q311H/M428L/N434Y F447 1.40E−08P257A/N286E/T307Q/M428L/N434Y F448 5.30E−08P257A/N286E/T307A/M428L/N434Y F449 5.70E−07S239K/M252Y/D270F/T307P/N325G/M428Y/N434Y F450 5.20E−07S239K/M252Y/T307P/L309E/N325G/M428Y/N434Y F451 1.00E−07P257S/T307A/M428L/N434Y F452 1.40E−07 P257M/T307A/M428L/N434Y F4537.80E−08 P257N/T307A/M428L/N434Y F454 9.60E−08 P257I/T307A/M428L/N434YF455 2.70E−08 P257V/T307Q/M428Y/N434Y F456 3.40E−08P257V/T307Q/M428F/N434Y F457 4.00E−08 S239K/P257V/V308P/M428L/N434Y F4581.50E−08 P257V/T307Q/V308P/N325G/M428L/N434Y F459 1.30E−08P257V/T307Q/V308P/Q311A/N325G/M428L/N434Y F460 4.70E−08P257V/T307A/V308P/N325G/M428L/N434Y F462 8.50E−08P257A/V308P/N325G/M428L/N434Y F463 1.30E−07P257A/T307A/V308P/M428L/N434Y F464 5.50E−08P257A/T307Q/V308P/M428L/N434Y F465 2.10E−08P257V/N286E/T307Q/N325G/M428L/N434Y F466 3.50E−07 T256E/P257V/N434Y F4675.70E−07 T256E/P257T/N434Y F468 5.70E−08 S239K/P257T/V308P/M428L/N434YF469 5.60E−08 P257T/V308P/N325G/M428L/N434Y F470 5.40E−08T256E/P257T/V308P/N325G/M428L/N434Y F471 6.60E−08P257T/V308P/N325G/E382A/M428L/N434Y F472 5.40E−08P257T/V308P/N325G/P387E/M428L/N434Y F473 4.50E−07P257T/V308P/L309P/N325G/M428L/N434Y F474 3.50E−07P257T/V308P/L309R/N325G/M428L/N434Y F475 4.30E−08T256E/P257V/T307Q/M428L/N434YTable 2-13 is a continuation of Table 2-12.

TABLE 2-13 F476 5.50E−08 P257V/T307Q/E382A/M428L/N434Y F477 4.30E−08P257V/T307Q/P387E/M428L/N434Y F480 3.90E−08 P257L/V308P/N434Y F4815.60E−08 P257T/T307Q/N434Y F482 7.00E−08 P257V/T307Q/N325G/N434Y F4835.70E−08 P257V/T307Q/Q311A/N434Y F484 6.20E−08 P257V/V305A/T307Q/N434YF485 9.70E−08 P257V/N286E/T307A/N434Y F486 3.40E−07P257V/T307Q/L309R/Q311H/M428L/N434Y F488 3.50E−08P257V/V308P/N325G/M428L/N434Y F490 7.50E−08S239K/P257V/V308P/Q311H/M428L/N434Y F492 9.80E−08P257V/V305A/T307A/N325G/M428L/N434Y F493 4.90E−07S239K/D270F/T307P/N325G/M428Y/N434Y F497 3.10E−06P257T/T307A/M428V/N434Y F498 1.30E−06 P257A/M428V/N434Y F499 5.20E−07P257A/T307A/M428V/N434Y F500 4.30E−08 P257S/T307Q/M428L/N434Y F5061.90E−07 P257V/N297A/T307Q/M428L/N434Y F507 5.10E−08P257V/N286A/T307Q/M428L/N434Y F508 1.10E−07P257V/T307Q/N315A/M428L/N434Y F509 5.80E−08P257V/T307Q/N384A/M428L/N434Y F510 5.30E−08P257V/T307Q/N389A/M428L/N434Y F511 4.20E−07 P257V/N434Y F512 5.80E−07P257T/N434Y F517 3.10E−07 P257V/N286E/N434Y F518 4.20E−07P257T/N286E/N434Y F519 2.60E−08 P257V/N286E/T307Q/N434Y F521 1.10E−08P257V/N286E/T307Q/M428Y/N434Y F523 2.60E−08P257V/V305A/T307Q/M428Y/N434Y F526 1.90E−08 P257T/T307Q/M428Y/N434Y F5279.40E−09 P257V/T307Q/V308P/N325G/M428Y/N434Y F529 2.50E−08P257T/T307Q/M428F/N434Y F533 1.20E−08 P257A/N286E/T307Q/M428F/N434Y F5341.20E−08 P257A/N286E/T307Q/M428Y/N434YTable 2-14 is a continuation of Table 2-13.

TABLE 2-14 F535 3.90E−08 T250A/P257V/T307Q/M428L/N434Y F538 9.90E−08T250F/P257V/T307Q/M428L/N434Y F541 6.00E−08T250I/P257V/T307Q/M428L/N434Y F544 3.10E−08T250M/P257V/T307Q/M428L/N434Y F549 5.40E−08T250S/P257V/T307Q/M428L/N434Y F550 5.90E−08T250V/P257V/T307Q/M428L/N434Y F551 1.20E−07T250W/P257V/T307Q/M428L/N434Y F552 1.10E−07T250Y/P257V/T307Q/M428L/N434Y F553 1.70E−07 M252Y/Q311A/N434Y F5542.80E−08 S239K/M252Y/S254T/V308P/N434Y F556 1.50E−06 M252Y/T307Q/Q311AF559 8.00E−08 M252Y/S254T/N286E/N434Y F560 2.80E−08M252Y/S254T/V308P/N434Y F561 1.40E−07 M252Y/S254T/T307A/N434Y F5628.30E−08 M252Y/S254T/T307Q/N434Y F563 1.30E−07 M252Y/S254T/Q311A/N434YF564 1.90E−07 M252Y/S254T/Q311H/N434Y F565 9.20E−08M252Y/S254T/T307A/Q311A/N434Y F566 6.10E−08M252Y/S254T/T307Q/Q311A/N434Y F567 2.20E−07 M252Y/S254T/M428I/N434Y F5681.10E−07 M252Y/T256E/T307A/Q311H/N434Y F569 2.00E−07M252Y/T256Q/T307A/Q311H/N434Y F570 1.30E−07M252Y/S254T/T307A/Q311H/N434Y F571 8.10E−08M252Y/N286E/T307A/Q311H/N434Y F572 1.00E−07M252Y/T307A/Q311H/M428I/N434Y F576 1.60E−06 M252Y/T256E/T307Q/Q311H F5771.30E−06 M252Y/N286E/T307A/Q311A F578 5.70E−07 M252Y/N286E/T307Q/Q311AF580 8.60E−07 M252Y/N286E/T307Q/Q311H F581 7.20E−08M252Y/T256E/N286E/N434Y F582 7.50E−07 S239K/M252Y/V308P F583 7.80E−07S239K/M252Y/V308P/E382A F584 6.30E−07 S239K/M252Y/T256E/V308P F5852.90E−07 S239K/M252Y/N286E/V308PTable 2-15 is a continuation of Table 2-14.

TABLE 2-15 F586 1.40E−07 S239K/M252Y/N286E/V308P/M428I F587 1.90E−07M252Y/N286E/M428L/N434Y F592 2.00E−07 M252Y/S254T/E382A/N434Y F5933.10E−08 S239K/M252Y/S254T/V308P/M428I/N434Y F594 1.60E−08S239K/M252Y/T256E/V308P/M428I/N434Y F595 1.80E−07S239K/M252Y/M428I/N434Y F596 4.00E−07 M252Y/D312A/E382A/M428Y/N434Y F5972.20E−07 M252Y/E382A/P387E/N434Y F598 1.40E−07 M252Y/D312A/P387E/N434YF599 5.20E−07 M252Y/P387E/M428Y/N434Y F600 2.80E−07M252Y/T256Q/E382A/N434Y F601 9.60E−09 M252Y/N286E/V308P/N434Y F608G236A/S239D/I332E F611 2.80E−07 M252Y/V305T/T307P/V308I/L309A/N434Y F6123.60E−07 M252Y/T307P/V308I/L309A/N434Y F613 S239D/A330L/I332E F616S239D/K326D/L328Y F617 7.40E−07 S239K/N434W F618 6.40E−07S239K/V308F/N434Y F619 3.10E−07 S239K/M252Y/N434Y F620 2.10E−07S239K/M252Y/S254T/N434Y F621 1.50E−07 S239K/M252Y/T307A/Q311H/N434Y F6223.50E−07 S239K/M252Y/T256Q/N434Y F623 1.80E−07 S239K/M252W/N434W F6241.40E−08 S239K/P257A/N286E/T307Q/M428L/N434Y F625 7.60E−08S239K/P257A/T307Q/M428L/N434Y F626 1.30E−06 V308P F629 3.90E−08M252Y/V279L/V308P/N434Y F630 3.70E−08 S239K/M252Y/V279L/V308P/N434Y F6332.40E−08 M252Y/V282D/V308P/N434Y F634 3.20E−08S239K/M252Y/V282D/V308P/N434Y F635 4.50E−08 M252Y/V284K/V308P/N434Y F6364.80E−08 S239K/M252Y/V284K/V308P/N434Y F637 1.50E−07M252Y/K288S/V308P/N434YTable 2-16 is a continuation of Table 2-15.

TABLE 2-16 F638 1.40E−07 S239K/M252Y/K288S/V308P/N434Y F639 2.70E−08M252Y/V308P/G385R/N434Y F640 3.60E−08 S239K/M252Y/V308P/G385R/N434Y F6413.00E−08 M252Y/V308P/Q386K/N434Y F642 3.00E−08S239K/M252Y/V308P/Q386K/N434Y F643 3.20E−08L235G/G236R/S239K/M252Y/V308P/N434Y F644 3.00E−08G236R/S239K/M252Y/V308P/N434Y F645 3.30E−08S239K/M252Y/V308P/L328R/N434Y F646 3.80E−08S239K/M252Y/N297A/V308P/N434Y F647 2.90E−08 P238D/M252Y/V308P/N434Y F648P238D F649 1.20E−07 S239K/M252Y/N286E/N434Y F650 1.70E−07S239K/M252Y/T256E/N434Y F651 1.80E−07 S239K/M252Y/Q311A/N434Y F6522.40E−07 P238D/M252Y/N434Y F654 3.20E−08 L235K/S239K/M252Y/V308P/N434YF655 3.40E−08 L235R/S239K/M252Y/V308P/N434Y F656 3.30E−08G237K/S239K/M252Y/V308P/N434Y F657 3.20E−08G237R/S239K/M252Y/V308P/N434Y F658 3.20E−08P238K/S239K/M252Y/V308P/N434Y F659 3.00E−08P238R/S239K/M252Y/V308P/N434Y F660 3.10E−08S239K/M252Y/V308P/P329K/N434Y F661 3.40E−08S239K/M252Y/V308P/P329R/N434Y F663 6.40E−09S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y F664 3.90E−08M252Y/N286A/V308P/N434Y F665 2.00E−08 M252Y/N286D/V308P/N434Y F6662.10E−08 M252Y/N286F/V308P/N434Y F667 3.00E−08 M252Y/N286G/V308P/N434YF668 4.00E−08 M252Y/N286H/V308P/N434Y F669 3.50E−08M252Y/N286I/V308P/N434Y F670 2.10E−07 M252Y/N286K/V308P/N434Y F6712.20E−08 M252Y/N286L/V308P/N434Y F672 2.40E−08 M252Y/N286M/V308P/N434YF673 2.30E−08 M252Y/N286P/V308P/N434YTable 2-17 is a continuation of Table 2-16.

TABLE 2-17 F674 3.20E−08 M252Y/N286Q/V308P/N434Y F675 5.10E−08M252Y/N286R/V308P/N434Y F676 3.20E−08 M252Y/N286S/V308P/N434Y F6774.70E−08 M252Y/N286T/V308P/N434Y F678 3.30E−08 M252Y/N286V/V308P/N434YF679 1.70E−08 M252Y/N286W/V308P/N434Y F680 1.50E−08M252Y/N286Y/V308P/N434Y F681 4.90E−08 M252Y/K288A/V308P/N434Y F6828.20E−08 M252Y/K288D/V308P/N434Y F683 5.00E−08 M252Y/K288E/V308P/N434YF684 5.10E−08 M252Y/K288F/V308P/N434Y F685 5.30E−08M252Y/K288G/V308P/N434Y F686 4.60E−08 M252Y/K288H/V308P/N434Y F6874.90E−08 M252Y/K288I/V308P/N434Y F688 2.80E−08 M252Y/K288L/V308P/N434YF689 4.10E−08 M252Y/K288M/V308P/N434Y F690 1.00E−07M252Y/K288N/V308P/N434Y F691 3.20E−07 M252Y/K288P/V308P/N434Y F6923.90E−08 M252Y/K288Q/V308P/N434Y F693 3.60E−08 M252Y/K288R/V308P/N434YF694 4.70E−08 M252Y/K288V/V308P/N434Y F695 4.00E−08M252Y/K288W/V308P/N434Y F696 4.40E−08 M252Y/K288Y/V308P/N434Y F6973.10E−08 S239K/M252Y/V308P/N325G/N434Y F698 2.20E−08M252Y/N286E/T307Q/Q311A/N434Y F699 2.30E−08S239K/M252Y/N286E/T307Q/Q311A/N434Y F700 5.20E−08M252Y/V308P/L328E/N434Y F705 7.10E−09 M252Y/N286E/V308P/M428I/N434Y F7061.80E−08 M252Y/N286E/T307Q/Q311A/M428I/N434Y F707 5.90E−09M252Y/N286E/T307Q/V308P/Q311A/N434Y F708 4.10E−09M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y F709 2.00E−08S239K/M252Y/N286E/T307Q/Q311A/M428I/N434Y F710 1.50E−08P238D/M252Y/N286E/T307Q/Q311A/M428I/N434Y F711 6.50E−08S239K/M252Y/T307Q/Q311A/N434YTable 2-18 is a continuation of Table 2-17.

TABLE 2-18 F712 6.00E−08 P238D/M252Y/T307Q/Q311A/N434Y F713 2.00E−08P238D/M252Y/N286E/T307Q/Q311A/N434Y F714 2.30E−07P238D/M252Y/N325S/N434Y F715 2.30E−07 P238D/M252Y/N325M/N434Y F7162.70E−07 P238D/M252Y/N325L/N434Y F717 2.60E−07 P238D/M252Y/N325I/N434YF718 2.80E−07 P238D/M252Y/Q295M/N434Y F719 7.40E−08P238D/M252Y/N325G/N434Y F720 2.40E−08 M252Y/T307Q/V308P/Q311A/N434Y F7211.50E−08 M252Y/T307Q/V308P/Q311A/M428I/N434Y F722 2.70E−07P238D/M252Y/A327G/N434Y F723 2.80E−07 P238D/M252Y/L328D/N434Y F7242.50E−07 P238D/M252Y/L328E/N434Y F725 4.20E−08L235K/G237R/S239K/M252Y/V308P/N434Y F726 3.70E−08L235K/P238K/S239K/M252Y/V308P/N434Y F729 9.20E−07 T307A/Q311A/N434Y F7306.00E−07 T307Q/Q311A/N434Y F731 8.50E−07 T307A/Q311H/N434Y F732 6.80E−07T307Q/Q311H/N434Y F733 3.20E−07 M252Y/L328E/N434Y F734 3.10E−07G236D/M252Y/L328E/N434Y F736 3.10E−07 M252Y/S267M/L328E/N434Y F7373.10E−07 M252Y/S267L/L328E/N434Y F738 3.50E−07 P238D/M252Y/T307P/N434YF739 2.20E−07 M252Y/T307P/Q311A/N434Y F740 2.90E−07M252Y/T307P/Q311H/N434Y F741 3.10E−07 P238D/T250A/M252Y/N434Y F7449.90E−07 P238D/T250F/M252Y/N434Y F745 6.60E−07 P238D/T250G/M252Y/N434YF746 6.00E−07 P238D/T250H/M252Y/N434Y F747 2.80E−07P238D/T250I/M252Y/N434Y F749 5.10E−07 P238D/T250L/M252Y/N434Y F7503.00E−07 P238D/T250M/M252Y/N434Y F751 5.30E−07 P238D/T250N/M252Y/N434YTable 2-19 is a continuation of Table 2-18.

TABLE 2-19 F753 1.80E−07 P238D/T250Q/M252Y/N434Y F755 3.50E−07P238D/T250S/M252Y/N434Y F756 3.70E−07 P238D/T250V/M252Y/N434Y F7571.20E−06 P238D/T250W/M252Y/N434Y F758 1.40E−06 P238D/T250Y/M252Y/N434YF759 L235K/S239K F760 L235R/S239K F761 1.10E−06 P238D/N434Y F7623.60E−08 L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y F763 3.50E−08L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y F764 6.30E−07P238D/T307Q/Q311A/N434Y F765 8.50E−08P238D/M252Y/T307Q/L309E/Q311A/N434Y F766 6.00E−07T307A/L309E/Q311A/N434Y F767 4.30E−07 T307Q/L309E/Q311A/N434Y F7686.40E−07 T307A/L309E/Q311H/N434Y F769 4.60E−07 T307Q/L309E/Q311H/N434YF770 3.00E−07 M252Y/T256A/N434Y F771 4.00E−07 M252Y/E272A/N434Y F7723.80E−07 M252Y/K274A/N434Y F773 3.90E−07 M252Y/V282A/N434Y F774 4.00E−07M252Y/N286A/N434Y F775 6.20E−07 M252Y/K338A/N434Y F776 3.90E−07M252Y/K340A/N434Y F777 3.90E−07 M252Y/E345A/N434Y F779 3.90E−07M252Y/N361A/N434Y F780 3.90E−07 M252Y/Q362A/N434Y F781 3.70E−07M252Y/S375A/N434Y F782 3.50E−07 M252Y/Y391A/N434Y F783 4.00E−07M252Y/D413A/N434Y F784 5.00E−07 M252Y/L309A/N434Y F785 7.40E−07M252Y/L309H/N434Y F786 2.80E−08 M252Y/S254T/N286E/T307Q/Q311A/N434Y F7878.80E−08 M252Y/S254T/T307Q/L309E/Q311A/N434Y F788 4.10E−07M252Y/N315A/N434YTable 2-20 is a continuation of Table 2-19.

TABLE 2-20 F789 1.50E−07 M252Y/N315D/N434Y F790 2.70E−07M252Y/N315E/N434Y F791 4.40E−07 M252Y/N315F/N434Y F792 4.40E−07M252Y/N315G/N434Y F793 3.30E−07 M252Y/N315I/N434Y F794 4.10E−07M252Y/N315K/N434Y F795 3.10E−07 M252Y/N315L/N434Y F796 3.40E−07M252Y/N315M/N434Y F798 3.50E−07 M252Y/N315Q/N434Y F799 4.10E−07M252Y/N315R/N434Y F800 3.80E−07 M252Y/N315S/N434Y F801 4.40E−07M252Y/N315T/N434Y F802 3.30E−07 M252Y/N315V/N434Y F803 3.60E−07M252Y/N315W/N434Y F804 4.00E−07 M252Y/N315Y/N434Y F805 3.00E−07M252Y/N325A/N434Y F806 3.10E−07 M252Y/N384A/N434Y F807 3.20E−07M252Y/N389A/N434Y F808 3.20E−07 M252Y/N389A/N390A/N434Y F809 2.20E−07M252Y/S254T/T256S/N434Y F810 2.20E−07 M252Y/A378V/N434Y F811 4.90E−07M252Y/E380S/N434Y F812 2.70E−07 M252Y/E382V/N434Y F813 2.80E−07M252Y/S424E/N434Y F814 1.20E−07 M252Y/N434Y/Y436I F815 5.50E−07M252Y/N434Y/T437R F816 3.60E−07 P238D/T250V/M252Y/T307P/N434Y F8179.80E−08 P238D/T250V/M252Y/T307Q/Q311A/N434Y F819 1.40E−07P238D/M252Y/N286E/N434Y F820 3.40E−07 L235K/S239K/M252Y/N434Y F8213.10E−07 L235R/S239K/M252Y/N434Y F822 1.10E−06P238D/T250Y/M252Y/W313Y/N434Y F823 1.10E−06P238D/T250Y/M252Y/W313F/N434Y F828 2.50E−06P238D/T250V/M252Y/I253V/N434YTable 2-21 is a continuation of Table 2-20.

TABLE 2-21 F831 1.60E−06 P238D/T250V/M252Y/R255A/N434Y F832 2.60E−06P238D/T250V/M252Y/R255D/N434Y F833 8.00E−07P238D/T250V/M252Y/R255E/N434Y F834 8.10E−07P238D/T250V/M252Y/R255F/N434Y F836 5.00E−07P238D/T250V/M252Y/R255H/N434Y F837 5.60E−07P238D/T250V/M252Y/R255I/N434Y F838 4.30E−07P238D/T250V/M252Y/R255K/N434Y F839 3.40E−07P238D/T250V/M252Y/R255L/N434Y F840 4.20E−07P238D/T250V/M252Y/R255M/N434Y F841 1.10E−06P238D/T250V/M252Y/R255N/N434Y F843 6.60E−07P238D/T250V/M252Y/R255Q/N434Y F844 1.30E−06P238D/T250V/M252Y/R255S/N434Y F847 3.40E−07P238D/T250V/M252Y/R255W/N434Y F848 8.30E−07P238D/T250V/M252Y/R255Y/N434Y F849 3.30E−07 M252Y/D280A/N434Y F8502.90E−07 M252Y/D280E/N434Y F852 3.30E−07 M252Y/D280G/N434Y F853 3.20E−07M252Y/D280H/N434Y F855 3.20E−07 M252Y/D280K/N434Y F858 3.20E−07M252Y/D280N/N434Y F860 3.30E−07 M252Y/D280Q/N434Y F861 3.20E−07M252Y/D280R/N434Y F862 3.00E−07 M252Y/D280S/N434Y F863 2.70E−07M252Y/D280T/N434Y F867 2.80E−07 M252Y/N384A/N389A/N434Y F868 2.00E−08G236A/S239D/M252Y/N286E/T307Q/Q311A/N434Y F869 G236A/S239D F870 7.30E−08L235K/S239K/M252Y/T307Q/Q311A/N434Y F871 7.10E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y F872 1.30E−07L235K/S239K/M252Y/N286E/N434Y F873 1.20E−07L235R/S239K/M252Y/N286E/N434Y F875 4.80E−07 M252Y/N434Y/Y436A F8778.30E−07 M252Y/N434Y/Y436E F878 1.90E−07 M252Y/N434Y/Y436FTable 2-22 is a continuation of Table 2-21.

TABLE 2-22 F879 9.20E−07 M252Y/N434Y/Y436G F880 3.90E−07M252Y/N434Y/Y436H F881 3.10E−07 M252Y/N434Y/Y436K F882 1.30E−07M252Y/N434Y/Y436L F883 2.10E−07 M252Y/N434Y/Y436M F884 4.00E−07M252Y/N434Y/Y436N F888 4.80E−07 M252Y/N434Y/Y436S F889 2.20E−07M252Y/N434Y/Y436T F890 1.10E−07 M252Y/N434Y/Y436V F891 1.70E−07M252Y/N434Y/Y436W F892 7.10E−08 M252Y/S254T/N434Y/Y436I F893 9.80E−08L235K/S239K/M252Y/N434Y/Y436I F894 9.20E−08L235R/S239K/M252Y/N434Y/Y436I F895 2.10E−08L235K/S239K/M252Y/N286E/T307Q/Q311A/N315E/ N434Y F896 2.00E−08L235R/S239K/M252Y/N286E/T307Q/Q311A/N315E/ N434Y F897 9.70E−08M252Y/N315D/N384A/N389A/N434Y F898 1.70E−07M252Y/N315E/N384A/N389A/N434Y F899 1.10E−07 M252Y/N315D/G316A/N434Y F9001.70E−07 M252Y/N315D/G316D/N434Y F901 1.30E−07 M252Y/N315D/G316E/N434YF902 2.20E−07 M252Y/N315D/G316F/N434Y F903 2.30E−07M252Y/N315D/G316H/N434Y F904 1.00E−07 M252Y/N315D/G316I/N434Y F9051.30E−07 M252Y/N315D/G316K/N434Y F906 1.50E−07 M252Y/N315D/G316L/N434YF907 1.30E−07 M252Y/N315D/G316M/N434Y F908 1.50E−07M252Y/N315D/G316N/N434Y F909 1.30E−07 M252Y/N315D/G316P/N434Y F9101.40E−07 M252Y/N315D/G316Q/N434Y F911 1.30E−07 M252Y/N315D/G316R/N434YF912 1.20E−07 M252Y/N315D/G316S/N434Y F913 1.10E−07M252Y/N315D/G316T/N434Y F914 1.50E−07 M252Y/N315D/G316V/N434Y F9152.30E−07 M252Y/N315D/G316W/N434YTable 2-23 is a continuation of Table 2-22.

TABLE 2-23 F917 2.50E−07 M252Y/N286S/N434Y F918 2.80E−07M252Y/D280E/N384A/N389A/N434Y F919 3.30E−07M252Y/D280G/N384A/N389A/N434Y F920 2.50E−07M252Y/N286S/N384A/N389A/N434Y F921 1.20E−07M252Y/N286E/N384A/N389A/N434Y F922 5.90E−08L235K/S239K/M252Y/N286E/N434Y/Y436I F923 6.00E−08L235R/S239K/M252Y/N286E/N434Y/Y436I F924 3.40E−08L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436I F925 3.20E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436I F926 1.10E−07L235K/S239K/M252Y/S254T/N434Y/Y436I F927 1.00E−07L235R/S239K/M252Y/S254T/N434Y/Y436I F928 2.90E−08M252Y/T307Q/Q311A/N434Y/Y436I F929 2.90E−08M252Y/S254T/T307Q/Q311A/N434Y/Y436I F930 1.40E−07P238D/T250V/M252Y/N286E/N434Y F931 1.20E−07P238D/T250V/M252Y/N434Y/Y436I F932 3.20E−07 T250V/M252Y/N434Y F9333.00E−07 L234R/P238D/T250V/M252Y/N434Y F934 3.10E−07G236K/P238D/T250V/M252Y/N434Y F935 3.20E−07G237K/P238D/T250V/M252Y/N434Y F936 3.20E−07G237R/P238D/T250V/M252Y/N434Y F937 3.10E−07P238D/S239K/T250V/M252Y/N434Y F938 1.60E−07L235K/S239K/M252Y/N434Y/Y436V F939 1.50E−07L235R/S239K/M252Y/N434Y/Y436V F940 1.50E−07P238D/T250V/M252Y/N434Y/Y436V F941 1.20E−08M252Y/N286E/T307Q/Q311A/N434Y/Y436V F942 4.20E−08L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436V F943 4.00E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V F944 1.70E−07T250V/M252Y/N434Y/Y436V F945 1.70E−08 T250V/M252Y/V308P/N434Y/Y436V F9464.30E−08 T250V/M252Y/T307Q/Q311A/N434Y/Y436V F947 1.10E−08T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F954 5.30E−07M252Y/N434Y/H435K/Y436V F957 7.70E−07 M252Y/N434Y/H435N/Y436V F9608.00E−07 M252Y/N434Y/H435R/Y436VTable 2-24 is a continuation of Table 2-23.

TABLE 2-24 F966 3.10E−07 M252Y/S254A/N434Y F970 2.50E−06M252Y/S254G/N434Y F971 2.60E−06 M252Y/S254H/N434Y F972 2.60E−07M252Y/S254I/N434Y F978 1.30E−06 M252Y/S254Q/N434Y F980 1.80E−07M252Y/S254V/N434Y F987 4.00E−08P238D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V F988 6.90E−08P238D/T250V/M252Y/N286E/N434Y/Y436V F989 1.40E−08L235R/S239K/M252Y/V308P/N434Y/Y436V F990 9.40E−09L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F991 1.30E−08L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y/Y436V F992 5.10E−08L235R/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V F993 3.80E−08M252Y/T307Q/Q311A/N434Y/Y436V F994 2.80E−07 M252Y/N325G/N434Y F9952.90E−07 L235R/P238D/S239K/M252Y/N434Y F996 1.30E−07L235R/P238D/S239K/M252Y/N434Y/Y436V F997 3.80E−07K248I/T250V/M252Y/N434Y/Y436V F998 8.50E−07K248Y/T250V/M252Y/N434Y/Y436V F999 2.10E−07T250V/M252Y/E258H/N434Y/Y436V F1005 N325G F1008 1.70E−07L235R/S239K/T250V/M252Y/N434Y/Y436V F1009 1.20E−08L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1010 1.90E−07L235R/S239K/M252Y/T307A/Q311H/N434Y F1011 4.50E−08T250V/M252Y/V308P/N434Y F1012 4.70E−08L235R/S239K/T250V/M252Y/V308P/N434Y F1013 3.00E−08T250V/M252Y/T307Q/V308P/Q311A/N434Y F1014 3.20E−08L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y F1015 2.20E−08L235R/S239K/M252Y/T307Q/V308P/Q311A/N434Y F1016 3.80E−09T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F1017 4.20E−09L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10183.20E−09 L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10193.40E−07 P238D/T250V/M252Y/N325G/N434Y F1020 8.50E−08P238D/T250V/M252Y/T307Q/Q311A/N325G/N434YTable 2-25 is a continuation of Table 2-24.

TABLE 2-25 F1021 3.30E−07 P238D/T250V/M252Y/N325A/N434Y F1022K326D/L328Y F1023 4.40E−08 S239D/T250V/M252Y/T307Q/Q311A/N434Y/Y436VF1024 4.00E−08 T250V/M252Y/T307Q/Q311A/K326D/L328Y/N434Y/Y436V F10253.60E−08 S239D/T250V/M252Y/T307Q/Q311A/K326D/L328Y/N434Y/Y436V F10268.40E−08 M252Y/T307A/Q311H/N434Y/Y436V F1027 8.60E−08L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V F1028 4.60E−08G236A/S239D/T250V/M252Y/T307Q/Q311A/N434Y/Y436V F1029 5.10E−08T250V/M252Y/T307Q/Q311A/I332E/N434Y/Y436V F1030 I332E F1031 5.30E−08G236A/S239D/T250V/M252Y/T307Q/Q311A/I332E/N434Y/Y436V F1032 4.30E−08P238D/T250V/M252Y/T307Q/Q311A/N325G/N434Y/Y436V F1033 1.00E−06P238D/N434W F1034 1.50E−08 L235K/S239K/M252Y/V308P/N434Y/Y436V F10351.00E−08 L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1036 1.40E−08L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y/Y436V F1037 6.10E−08L235K/S239K/M252Y/T307Q/Q311A/M428I/N434Y/Y436V F1038 2.80E−07L235K/P238D/S239K/M252Y/N434Y F1039 1.30E−07L235K/P238D/S239K/M252Y/N434Y/Y436V F1040 2.00E−07L235K/S239K/T250V/M252Y/N434Y/Y436V F1041 1.40E−08L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1042 2.00E−07L235K/S239K/M252Y/T307A/Q311H/N434Y F1043 5.20E−08L235K/S239K/T250V/M252Y/V308P/N434Y F1044 3.50E−08L235K/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y F1045 2.50E−08L235K/S239K/M252Y/T307Q/V308P/Q311A/N434Y F1046 4.50E−09L235K/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10473.40E−09 L235K/S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y/Y436V F10489.90E−08 L235K/S239K/M252Y/T307A/Q311H/N434Y/Y436V F1050 3.50E−09T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V F1051 3.90E−09L235R/S239K/T250V/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436V F10523.20E−09 L235R/S239K/M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y/Y436VTable 2-26 is a continuation of Table 2-25.

TABLE 2-26 F1053 4.23E−08 L235R/S239K/T250V/M252Y/T307Q/Q311A/N434Y/Y436V F1058 1.31E−07 M252Y/Q386E/N434Y/Y436V F1059 1.39E−07M252Y/Q386R/N434Y/Y436V F1060 1.43E−07 M252Y/Q386S/N434Y/Y436V F10611.19E−07 M252Y/P387E/N434Y/Y436V F1062  1.2E−07 M252Y/P387R/N434Y/Y436VF1063 1.43E−07 M252Y/P387S/N434Y/Y436V F1064 1.32E−07M252Y/V422E/N434Y/Y436V F1065 1.38E−07 M252Y/V422R/N434Y/Y436V F10661.45E−07 M252Y/V422S/N434Y/Y436V F1067 1.26E−07 M252Y/S424E/N434Y/Y436VF1068 1.69E−07 M252Y/S424R/N434Y/Y436V F1069 1.39E−07M252Y/N434Y/Y436V/Q438E F1070 1.73E−07 M252Y/N434Y/Y436V/Q438R F10711.24E−07 M252Y/N434Y/Y436V/Q438S F1072 1.35E−07 M252Y/N434Y/Y436V/S440EF1073 1.34E−07 M252Y/N434Y/Y436V/S440R F1074 1.32E−07S239D/M252Y/N434Y/Y436V F1075  1.4E−07 M252Y/K326D/L328Y/N434Y/Y436VF1076 1.27E−07 S239D/M252Y/K326D/L328Y/N434Y/Y436V F1077 2.03E−06K248N/M252Y/N434Y F1078  4.7E−07 M252Y/E380N/E382S/N434Y F1079 3.44E−07N252Y/E382N/N384S/N434Y F1080 3.19E−07 M252Y/S424N/N434Y F1081  6.2E−07M252Y/N434Y/Y436N/Q438T F1082 2.76E−07 M252Y/N434Y/Q438N F1083 3.45E−07M252Y/N434Y/S440N F1094  2.6E−07 M252Y/N434Y/S442N F1095 2.86E−07M252Y/S383N/G385S/N434Y F1096 2.72E−07 M252Y/Q386T/N434Y F1097 2.82E−07M252Y/G385N/P387S/N434Y F1098 2.58E−07 S239D/M252Y/N434Y F1099 2.57E−07M252Y/K326D/L328Y/N434Y F1100 2.41E−07 S239D/M252Y/K326D/L328Y/N434YF1101 6.59E−08 S239D/M2S2Y/T307Q/Q311A/N434Y F1102 6.46E−08M252Y/T307Q/Q311A/K326D/L328Y/N434Y F1103 6.11E−08S239D/M252Y/T307Q/Q311A/K326D/ L328Y/N434Y F1104 1.77E−07M252Y/V422E/S424R/N434Y/Y436V F1105 1.54E−07M252Y/V422S/S424R/N434Y/Y436V F1106 1.42E−07M252Y/N434Y/Y436V/Q438R/S440E F1107 1.23E−07 M252Y/V422D/N434Y/Y436VTable 2-27 is a continuation of Table 2-26.

TABLE 2-27 F1108 1.26E−07 M252Y/V422K/N434Y/Y436V F1109 1.27E−07M252Y/V422T/N434Y/Y436V F1110 1.33E−07 M252Y/V422Q/N434Y/Y436V F11111.65E−07 M252Y/S424K/N434Y/Y436V F1112 1.23E−07 M252Y/N434Y/Y436V/Q438KF1113 1.18E−07 M252Y/N434Y/Y436V/S440D F1114 1.31E−07M252Y/N434Y/Y436V/S440Q F1115 1.35E−07 M252Y/S424N/N434Y/Y436V F11167.44E−08 M252Y/T307Q/Q311A/S424N/N434Y F1117 4.87E−08T250V/M252Y/T307Q/Q311A/S424N/N434Y/Y436V F1118 1.32E−08T250V/M252Y/T307Q/V308P/Q311A/S424N/N434Y/Y436V F1119 1.03E−08T250V/M252Y/T307Q/V308P/Q311A/V422E/N434Y/Y436V F1120 1.04E−08T250V/M252Y/T307Q/V308P/Q311A/S424R/N434Y/Y436V F1121 1.04E−08T250V/M252Y/T307Q/V308P/Q311A/V422E/S424R/N434Y/Y436V F1122 1.37E−08T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R F1123 9.55E−09T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/S440E F1124 1.22E−08T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R/S440E F1125 5.18E−08M252Y/T307Q/N434Y/Y436V F1126 8.95E−08 M252Y/T307A/N434Y/Y436V F11277.94E−08 M252Y/Q311A/N434Y/Y436V F1128 1.17E−07 M252Y/Q311H/N434Y/Y436VF1129 4.48E−08 M252Y/T307Q/Q311H/N434Y/Y436V F1130 5.54E−08M252Y/T307A/Q311A/N434Y/Y436V F1131 1.29E−07L235R/S239K/M252Y/V422E/N434Y/Y436V F1132  1.4E−07L235K/S239K/M252Y/V422S/N434Y/Y436V F1133 1.58E−07L235R/S239K/M252Y/S424R/N434Y/Y436V F1134 1.66E−07L235R/S239K/M252Y/N434Y/Y436V/Q438R F1135 1.26E−07L235R/S239K/M252Y/N434Y/Y436V/S440E F1136 1.63E−07L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V F1137 1.58E−07L235R/S239K/M252Y/V422S/S424R/N434Y/Y436V F1138 1.65E−07L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440E F1139 1.52E−07L235R/S239K/M252Y/S424N/N434Y/Y436V F1140 1.62E−07M252Y/V422E/S424R/N434Y/Y436V/Q438R/S440E F1141 1.77E−07M252Y/V422S/S424R/N434Y/Y436V/Q438R/S440E F1142 1.87E−07L235R/S239K/M252Y/V422E/S424R/N434Y/Y436V/Q438R/S440E F1143 1.98E−07L235R/S239K/M252Y/V422S/S424R/N434Y/Y436V/Q438R/S440E F1144 1.44E−08L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V/Q438R/S440E F11455.23E−08 T250V/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E F1146 6.24E−08L235R/S239K/T250V/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E F11477.19E−08 M252Y/T307Q/Q311A/N434Y/Q438R/S440ETable 2-28 is a continuation of Table 2-27.

TABLE 2-28 F1148 7.63E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y/Q438R/S440E F1151 2.51E−07L235R/S239K/M252Y/S424N/N434Y F1152 7.38E−08L233R/S239K/M252Y/T307Q/Q311A/S424N/N434Y F1153 4.85E−08L235R/S239K/T250V/M252Y/T307Q/Q311A/S424N/N434Y/Y436V F1154 1.34E−08L235R/S239K/T250V/M252Y/T307Q/V308P/Q311A/S434N/M34Y/Y436V F11572.09E−07 M252Y/N434Y/Q438R/S440E F1158 2.44E−07L235R/S239K/M252Y/N434Y/Q438R/S440E F1159 4.79E−07 S424N/N434W F11602.88E−07 V308F/S424N/N434Y F1161 1.07E−06 I332V/S424N/N434Y F11623.43E−07 P238D/T250Y/M252Y/N434Y/Y436V F1163 1.54E−07P238D/T250Y/M252Y/T307Q/Q311A/N434Y F1164 6.96E−08P238D/T250Y/M252Y/T307Q/Q311A/N434Y/Y436V F1165 1.63E−08P238D/T250Y/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1174  4.9E−07P257I/N434H F1176 1.98E−06 V308F F1178 8.72E−07 V259I/V308F/M428L F11831.28E−06 E380A/M428L/N434S F1184   1E−06 T307A/M428L/N434S F11859.17E−07 T307A/E380A/M428L/N431S F1188 1.72E−06 T307A/E380A/N434H F11891.57E−07 M252Y/H433D/N434Y/Y436V/Q138R/S440E F1190  2.4E−07M252Y/H433E/N434Y/Y436V/Q438R/S440E F1191 2.11E−07M252Y/N434Y/Y436V/T437A/Q438R/S440E F1192 1.27E−07M252Y/N434Y/Y436V/T437G/Q438R/S440E F1194 1.55E−07M252Y/N434Y/Y436V/Q438R/K439D/S440E F1195 1.76E−07M252Y/N434Y/Y436V/Q438R/S440E/L441A F1196 1.51E−07M252Y/N434Y/Y436V/Q438R/S440E/L441E F1197 9.46E−08M252Y/S254T/N434Y/Y436V/Q438K/S440E F1198 7.83E−08M252Y/T256E/N434Y/Y436V/Q438R/S440E F1199 6.25E−08M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1200 1.26E−07T250V/M252Y/S254T/N434Y/Y436V/Q438R/S440E F1201 1.07E−07T250V/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1202 8.81E−08T250V/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1203 1.52E−07M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1204 1.18E−07M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E F1205 1.98E−07T250V/M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1206 1.69E−07T250V/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E F1207 1.11E−06I332E/M428L/N434S F1208 5.71E−07 L251A/M252Y/N434Y/Y436V F1211 1.23E−06L251H/M252Y/N434Y/Y436VTable 2-29 is a continuation of Table 2-28.

TABLE 2-29 F1213 6.33E−07 L251N/M252Y/N434Y/Y436V F1216 1.16E−06L251S/M252Y/N434Y/Y436V F1217 1.14E−06 L251T/M252Y/N434Y/Y436V F12182.51E−07 L251V/M252Y/N434Y/Y436V F1229 2.81E−06 M252Y/I253V/N434Y/Y436VF1230 1.12E−07 M252Y/N434Y/Y436V/Q438R/S440D F1231 9.73E−08M252Y/N434Y/Y436V/Q438K/S440E F1232 9.79E−08M252Y/N434Y/Y436V/Q438K/S440D F1243 1.25E−07L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E F1244 1.02E−07L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1245  8.2E−08L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F1246 1.73E−07L235R/S239K/T250V/M252Y/S254T/N434Y/Y436V/Q438R/S440E F1247 1.45E−07L235R/S239K/T250V/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1248  1.2E−07L235R/S239K/T250V/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F12492.06E−07 L235R/S239K/M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1250 1.66E−07L235R/S239K/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E F1251 2.77E−07L235R/S239K/T250V/M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1252 2.33E−07L235R/S239K/T250V/M252Y/S254T/T256Q/N434Y/Y436V/Q438R/S440E F12531.12E−07 L235R/S239K/M252Y/T307A/N434Y/Y436V/Q438R/S440E F1254 6.42E−08L235R/S239K/M252Y/T307Q/N434Y/Y436V/Q438R/S440E F1255 1.11E−07L235R/S239K/M252Y/Q311A/N434Y/Y436V/Q438R/S440E F1256 1.56E−07L235R/S239K/M252Y/Q311H/N434Y/Y436V/Q438R/S440E F1257 7.81E−08L235R/S239K/M252Y/T307A/Q311A/N434Y/Y436V/Q438R/S440E F1258 1.05E−07L235R/S239K/M252Y/T307A/Q311H/N434Y/Y436V/Q438R/S440E F1259 4.46E−08L235R/S239K/M252Y/T307Q/Q311A/N434Y/Y436V/Q438R/S440E F1260 6.53E−08L235R/S239K/M252Y/T307Q/Q311H/N434Y/Y436V/Q438R/S440E F1261 1.35E−07L235R/S239K/M252Y/N434Y/Y436V/Q438R/S440D F1262 1.26E−07L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440E F1263 1.24E−07L235R/S239K/M252Y/N434Y/Y436V/Q438K/S440D F1264 1.27E−07L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438R/S440E F1265 1.57E−07L235R/S239K/M252Y/T256G/N434Y/Y436V/Q438R/S440E F1266 9.99E−08L235R/S239K/M252Y/T256N/N434Y/Y436V/Q438R/S440E F1267  1.5E−07L235R/S239K/M252Y/S254A/N434Y/Y436V/Q438R/S440E F1268   2E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440E F1269 1.69E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440D F1270 1.18E−07L235R/S239K/M252Y/S254A/N434Y/Y436V/Q438K/S440D F1271 2.05E−07L235R/S239K/M252Y/S254A/H433D/N434Y/Y436V/Q438R/S440E F1272 1.71E−07L235R/S239K/M252Y/S254A/H433D/N434Y/Y436V/Q438K/S440D F1273 1.53E−07L235R/S239K/M252Y/T256Q/N434Y/Y436V/Q438K/S440D F1274 2.48E−07L235R/S239K/M252Y/T256Q/H433D/N434Y/Y436V/Q438R/S440E F1275 2.09E−07L235R/S239K/M252Y/T256Q/H433D/N434Y/Y436V/Q438K/S440DTable 2-30 is a continuation of Table 2-29.

TABLE 2-30 F1276 1.02E−07L235R/S239K/M252Y/T256A/N434Y/Y436V/Q438K/S440D F1277 1.69E−07L235K/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438R/S440E F1278  1.4E−07L235R/S239K/M252Y/T256A/H433D/N434Y/Y436V/Q438K/S440D F1279 1.23E−07L235R/S239K/M252Y/T256G/N436Y/Y436V/Q438K/S440D F1280 2.09E−07L235R/S239K/M252Y/T256G/H433D/N434Y/Y436V/Q438R/S440E F1281 1.74E−07L235R/S239K/M252Y/T256G/H433D/N434Y/Y436V/Q438K/S440D F1282 7.69E−08L235P/S239K/M252Y/T256N/N434Y/Y436V/Q438K/S440D F1283 1.34E−07L235R/S239K/M252Y/T256N/H433D/N434Y/V436V/Q438R/S440E F1284 1.12E−07L235R/S339K/M252Y/T256N/H433D/N434Y/Y436V/Q438K/S440D F1285 9.36E−08L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440D F1286 1.57E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440E F1287  1.5E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440D F1288 7.95E−08L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440D F1289 1.33E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438R/S440E F1290 1.11E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438K/S440D F1291 1.51E−07L235R/S239K/M252Y/H433D/N434Y/Y436V F1292 4.24E−07L235R/S239K/H433D/N434W/Y436V/Q438R/S440E F1293 1.61E−07L235R/S239K/M252Y/T256E/N434Y/Q438R/S440E F1294   2E−07L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438R/S440E F1295 9.84E−08L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438R/S440E F1296 2.27E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Q438R/S440E F1297  2.5E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438R/S440E F1298 1.47E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438R/S440E F1299  1.5E−07L235R/S239K/M252Y/T256E/N434Y/Q438K/S440D F1300 1.63E−07L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440D F1301  8.3E−08L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438K/S440D F1302 2.15E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Q438K/S440D F1303  2.1E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438K/S440D F1304 1.24E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438K/S440D F1305 2.05E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438R/S440D F1306 1.92E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438K/S440E F1307 1.44E−07L235R/S239K/M252Y/V422A/S424A/N434Y/Y436V F1308 2.06E−07L235R/S239K/M252Y/V422L/S424L/N434Y/Y436V F1309 1.26E−07L235R/S239K/M252Y/N434Y/Y436V/Q438A/S440A F1310 2.28E−07L235R/S239K/M252Y/N434Y/Y436V/Q438L/S440L F1311 1.69E−07L235R/S239K/M252Y/V422A/S424A/H433D/N434Y/Y436V F1312 1.79E−07L235R/S239K/M252Y/V422L/S424L/H433D/N434Y/Y436V F1313 1.77E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438A/S440A F1314 2.27E−07L235R/S239K/M252Y/H433D/N434Y/Y436V/Q438L/S440L F1315 1.52E−07G237K/S239K/M252Y/N434Y/Y436V F1316 1.49E−07G337R/S239K/M252Y/N434Y/Y436VTable 2-31 is a continuation of Table 2-30.

TABLE 2-31 F1317 1.38E−07 S239K/M252Y/P329K/N434Y/Y436V F1318 1.43E−07S239K/M252Y/P329R/N434Y/Y436V F1319 2.67E−07 M252Y/L328Y/N434Y F13201.22E−07 L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440D F1321 1.03E−07L235R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440E F1322  1.6E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438R/S440D F1323 1.49E−07L235R/S239K/M252Y/S254T/H433D/N434Y/Y436V/Q438K/S440E F1324 1.32E−07L234A/L235A/M252Y/N434Y/Y436V F1325 2.13E−07L234A/L235A/M252Y/N297A/N434Y/Y436V F1326 1.09E−08L234A/L235A/T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F1327 1.41E−08L234A/L235A/T250V/M252Y/N297A/T307Q/V308P/Q311A/N434Y/Y436V F13281.52E−07 L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438R/S440E F1329 1.29E−07L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438R/S440E F1330 1.03E−07L235R/G236R/S239K/M252Y/T256E/N434Y/Y436V/Q438R/S440E F1331 7.75E−08L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438R/S440E F13331.23E−07 L235R/G236R/S239K/M252Y/N434Y/Y436V F1334 1.04E−07L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438K/S440D F1335 8.78E−08L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440D F1336 7.18E−08L235R/G236R/S239K/M232Y/T256E/N434Y/Y436V/Q438K/S440D F1337 7.41E−08L235R/S239K/M252Y/T256E/N434Y/Y436V/Q438K/S440E F1338 1.04E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436V/Q438K/S440E F1339 2.51E−07L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436T/Q438K/S440E F13405.58E−08 L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E F13413.22E−07 L235R/S339K/M252Y/S254T/N434Y/Y436T/Q438K/S440E F1342 2.51E−07L235R/S239K/M252Y/T256E/N434Y/Y436T/Q438K/S440E F1343 2.01E−07L235R/S239K/M252K/S254T/T256E/N434Y/Y436T/Q438K/S440E F1344 3.96E−07L235R/S239K/M252Y/N434Y/Y436T/Q438K/S440E F1345 1.05E−07L235R/G236R/S239K/M252Y/N434Y/Y436V/Q438K/S440E F1346 8.59E−08L235R/G236R/S239K/M252Y/S254T/N434Y/Y436V/Q438K/S440E F1347 7.14E−08L235R/G236R/S339K/M252Y/T256E/N434Y/Y436V/Q438K/S440E F1348 5.52E−08L235R/G236R/S239K/M252Y/S254T/T256E/N434Y/Y436V/Q438K/S440E F13493.36E−07 L235R/S239K/M252Y/N434Y/Y436T/Q438R/S440E F1350 1.18E−07L235R/S239K/M252Y/N434Y/Y436F/Q438K/S440E F1351 1.62E−07L235R/S239K/M252Y/N434Y/Y436F/Q438R/S440E F1352 3.93E−07L235R/S239K/M252Y/H433D/N434Y/Y436T/Q438K/S440E F1353 4.33E−07L235R/S239K/M252Y/H433D/N434Y/Y436T/Q438R/S440E F1354 2.29E−07L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438K/S440E F1355 2.47E−07L235R/S239K/M252Y/H433D/N434Y/Y436F/Q438R/S440E F1356 1.58E−07G236R/M252Y/L328K/N434Y/Y436V F1357 2.81E−07L235R/S239K/M252Y/S254T/N434Y/Y436T/Q438R/S440E F1358 9.07E−08L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438K/S440ETable 2-32 is a continuation of Table 2-31.

TABLE 2-32 F1359 1.28E−07L235R/S239K/M252Y/S254T/N434Y/Y436F/Q438R/S440E F1360 3.12E−07L235R/S239K/M252Y/S254T/H443D/N434Y/Y436T/Q438K/S440E F1361 3.52E−07L235R/S239K/M252Y/S254T/H443D/N434Y/Y436F/Q438R/S440E F1362 1.41E−07L235R/S239K/M252Y/S254T/H443D/N434Y/Y436F/Q438K/S440E F1363  1.9E−07L235R/S239K/M252Y/S254T/H443D/N434Y/Y436F/Q438R/S440E F1364 7.49E−08L235R/S239K/M252Y/T256E/N434Y/Y436F/Q438K/S440E F1365 3.14E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436T/Q438K/S440E F1366 1.17E−07L235R/S239K/M252Y/T256E/H433D/N434Y/Y436F/Q438K/S440E F1367 1.79E−07L235R/S239K/M252Y/S254T/T256E/N434Y/Y436T/Q438R/S440E F1368 5.49E−08L235R/S239K/M252Y/S254T/T256E/N434Y/Y436F/Q438K/S440E F1369  7.6E−08L235R/S239K/M252Y/S254T/T256E/N434Y/Y436F/Q438R/S440E F1370 9.14E−08L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438K/S440E F13711.09E−07 L235R/S239K/M252Y/S254T/T256E/H433D/N434Y/Y436V/Q438R/S440EF1372 2.28E−07L235R/S239K/M252Y/S254T/T256E/H433D/N343Y/Y436T/Q438R/S440E F13738.67E−08 L235R/S239K/M252Y/S254T/T256E/H433D/N343Y/Y436F/Q438K/S440EF1374  1.2E−07L235R/S239K/M252Y/S254T/T256E/H433D/N343Y/Y436F/Q438R/S440E F13751.03E−07 L235R/S239K/M252Y/S254T/N434Y/Y436V F1376 9.09E−08L235R/S239K/M252Y/S254T/T256E/N434Y/Y436V F1377 8.27E−08L235R/S239K/M252Y/T256E/N434Y/Y436V F1378 3.61E−07L235R/S239K/M252Y/N434Y/Y436T F1379 2.85E−07L235R/S239K/M252Y/N434Y/Y436F F1410 1.90E−06 V308P/1332V F1411 1.70E−07V308P/1332V/M428L/N434S F1413 3.70E−08L235R/S239K/M252Y/S252Y/S254T/T256E/T307Q/Q311A/H433D/N434Y/Y436V/Q438K/S440E F1414 5.60E−08L235R/S239K/M252Y/S252Y/S254T/T256E/T307Q/H433D/N434Y/Y436V/Q438K/S440EF1415 5.90E−08L235R/S239K/M252Y/S252Y/S254T/T256E/Q311A/H433D/N434Y/Y436V/Q438K/S440EF1416 1.30E−08L235R/S239K/M252Y/S252Y/S254T/T256E/V308P/H433D/N434Y/Y436V/Q438K/S440EF1417 5.90E−08L235R/S239K/M252Y/S252Y/S254T/T256E/H433D/N434W/Y436V/Q438K/S440E F14187.50E−08L235R/S239K/M252Y/S252Y/S254T/T256E/H433D/N434W/Y436V/Q438R/S440E F14191.50E−07 L235R/S239K/M252Y/H433D/N434W/Y436V/Q438R/S440E F1420 1.30E−07L235R/S239K/M252Y/H433D/N434W/Y436V/Q438K/S440E F1421 3.20E−08V308P/M428L/N434W F1422 1.90E−08L235R/S239K/M252Y/T256E/V308P/H433D/N434Y/Y436V/Q438R/S440E F14231.60E−08L235R/S239K/M252Y/T256E/V302D/V308P/H433D/N434Y/Y436V/Q438R/S440E F14241.60E−08L235R/S239K/M252Y/T256E/V302E/V308P/H433D/N434Y/Y436V/Q438R/S440ETable 2-33 is a continuation of Table 2-32.

TABLE 2-33 F1425 1.90E−08L235R/S239K/M252Y/T256E/V303D/V308P/H433D/N434Y/Y436V/Q438R/S440E F14261.80E−08L235R/S239K/M252Y/T256E/V303E/V308P/H433D/N434Y/Y436V/Q438R/S440E F14281.50E−08L235R/S239K/M252Y/T256E/S304E/V308P/H433D/N434Y/Y436V/Q438R/S440E F14303.10E−08L235R/S239K/M252Y/T256E/V305E/V308P/H433D/N434Y/Y436V/Q438R/S440E F14334.50E−08L235R/S239K/M252Y/T256E/T307D/V308P/H433D/N434Y/Y436V/Q438R/S440E F14343.60E−08L235R/S239K/M252Y/T256E/T307E/V308P/H433D/N434Y/Y436V/Q438R/S440E

Heterocomplex Comprising the Four Molecules Including Two Molecules ofFcRn and One Molecule of Activating Fcγ Receptor

Crystallographic studies on FcRn with IgG antibodies demonstrated thatan FcRn-IgG complex is composed of one molecule of IgG for two moleculesof FcRn, and the two molecules are thought to bind around the interfaceof the CH2 and CH3 domains located on both sides of the IgG Fc region(Burmeister et al. (Nature (1994) 372, 336-343)). Meanwhile, asdemonstrated in Example 3 of PCT/JP2012/058603, the antibody Fc regionwas demonstrated to be able to form a complex comprising the fourmolecules including two molecules of FcRn and one molecule of activatingFcγ receptor (PCT/JP2012/058603). This heterocomplex formation is aphenomenon which was revealed as a result of analyzing the properties ofantigen-binding molecules containing an Fc region having an FcRn-bindingactivity under a neutral pH range condition.

While the present invention is not bound to a particular principle, itcan be considered that antigen-binding molecules administered in vivoproduce the effects described below on the in vivo pharmacokinetics(plasma retention) of the antigen-binding molecules and an immuneresponse (immunogenicity) to the administered antigen-binding molecules,as a result of the formation of heterocomplexes containing the fourmolecules including the Fc region contained in the antigen-bindingmolecules, two molecules of FcRn, and one molecule of activating Fcγreceptor. In addition to the various types of activating Fcγ receptors,FcRn is expressed on immune cells. It is suggested that the formation ofsuch tetrameric complexes on immune cells by antigen-binding moleculespromotes incorporation of antigen-binding molecules into immune cells byincreasing affinity toward immune cells and by causing association ofintracellular domains to enhance the internalization signal. The samealso applies to antigen-presenting cells and the possibility thatantigen binding-molecules are likely to be incorporated intoantigen-presenting cells by formation of tetrameric complexes on thecell membrane of antigen-presenting cells. In general, antigen-bindingmolecules incorporated into antigen-presenting cells are degraded in thelysosomes of the antigen-presenting cells and are presented to T cells.As a result, plasma retention of antigen-binding molecules may beworsened because incorporation of antigen-binding molecules intoantigen-presenting cells is promoted by the formation of theabove-described tetrameric complexes on the cell membrane of theantigen-presenting cells. Similarly, an immune response may be induced(aggravated).

For this reason, it is conceivable that when an antigen-binding moleculehaving lowered ability to form such tetrameric complexes is administeredin vivo, plasma retention of the antigen-binding molecules wouldimprove, and induction of in vivo immune response would be suppressed.Preferred embodiments of such antigen-binding molecules which inhibitthe formation of these complexes on immune cells includingantigen-presenting cells are, for example, the three embodimentsdescribed below.

Antigen-Binding Molecules which Inhibit the Formation of HeterocomplexesEmbodiment 1 An Antigen-Binding Molecule Containing an Fc Region HavingFcRn-Binding Activity Under a Neutral pH Range Condition and WhoseBinding Activity Toward Activating FcγR is Lower than the BindingActivity of a Native Fc Region Toward Activating FcγR

The antigen-binding molecule of Embodiment 1 forms a trimeric complex bybinding to two molecules of FcRn; however, it does not form any complexcontaining activating FcγR. An Fc region whose binding activity towardactivating FcγR is lower than the binding activity of a native Fc regiontoward activating FcγR can be prepared by altering the amino acids ofthe native Fc region as described above. Whether the binding activitytoward activating FcγR of the altered Fc region is lower than thebinding activity toward activating FcγR of the native Fc region can beappropriately tested using the methods described in the section “BindingActivity” above.

Preferred activating Fcγ receptors include FcγRI (CD64) which includesFcγRIa, FcγRIb, and FcγRIc; FcγRIIa (including allotypes R131 and H131);and FcγRIII (CD16) which includes isoforms FcγRIIIa (including allotypesV158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 andFcγRIIIb-NA2).

Herein, “a binding activity of the Fc region variant toward anactivating Fcγ receptor is lower than the binding activity of the nativeFc region toward an activating Fcγ receptor” means that the bindingactivity of the Fc region variant toward any of the human Fcγ receptors(FcγRI, FcγRIIa, FcγRIIIa, and/or FcγRIIIb) is lower than the bindingactivity of the native Fc region toward these human Fcγ receptors. Forexample, it means that based on an above-described analytical method,the binding activity of the antigen-binding molecule containing an Fcregion variant as compared to the binding activity of an antigen-bindingmolecule containing a native Fc region as a control is 95% or less,preferably 90% or less, 85% or less, 80% or less, 75% or less, andparticularly preferably 70% or less, 65% or less, 60% or less, 55% orless, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less,25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% orless, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% orless, or 1% or less. As a native Fc region, a starting Fc region may beused, and Fc regions of wild-type antibodies of different isotypes mayalso be used.

Meanwhile, the binding activity of the native form toward an activatingFcγR is preferably a binding activity toward the Fcγ receptor for humanIgG1. Other than performing the above-described alterations, bindingactivity toward the Fcγ receptor can be lowered by changing the isotypeto human IgG2, human IgG3, or human IgG4. Alternatively, besides byperforming the above-described alterations, the binding activity towardan Fcγ receptor can also be lowered by expressing the antigen-bindingmolecule containing an Fc region having a binding activity toward theFcγ receptor in hosts that do not add sugar chains such as Escherichiacoli.

For the antigen-binding molecule containing a control Fc region, anantigen-binding molecule having an Fc region of a monoclonal IgGantibody may be appropriately used. The structures of such Fc regionsare shown in SEQ ID NO: 5 (A is added to the N terminus of RefSeqAccession No. AAC82527.1), SEQ ID NO: 6 (A is added to the N terminus ofRefSeq Accession No. AAB59393.1), SEQ ID NO: 7 (RefSeq Accession No.CAA27268.1), and SEQ ID NO: 8 (A is added to the N terminus of RefSeqAccession No. AAB59394.1). Further, when an antigen-binding moleculecontaining an Fc region of a particular antibody isotype is used as thetest substance, effect on the binding activity of the antigen-bindingmolecule containing the Fc region toward an Fcγ receptor is tested byusing the antigen-binding molecule having an Fc region of a monoclonalIgG antibody of a particular isotype as a control. In this way,antigen-binding molecules containing an Fc region whose binding activitytoward the Fcγ receptor was demonstrated to be high are suitablyselected.

In a non-limiting embodiment of the present invention, preferredexamples of Fc regions whose binding activity toward an activating FcγRis lower than the binding activity of the native Fc region toward anactivating FcγR include Fc regions with alteration of one or more aminoacids at any of positions 234, 235, 236, 237, 238, 239, 270, 297, 298,325, 328, and 329 as indicated by EU numbering in the amino acids of anabove-described Fc region to be different from those of the native Fcregion. The alterations in the Fc region are not limited to the aboveexample, and they may be, for example, modifications such asdeglycosylation (N297A and N297Q), IgG1-L234A/L235A,IgG1-A325A/A330S/P331S, IgG1-C226S/C229S,IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S,IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268QN309L/A330S/A331S,IgG4-L235A/G237A/E318A, and IgG4-L236E described in Cur. Opin. inBiotech. (2009) 20 (6), 685-691; alterations such as G236R/L328R,L235G/G236R, N325A/L328R, and N325L/L328R described in WO 2008/092117;amino acid insertions at positions 233, 234, 235, and 237 according toEU numbering; and alterations at the positions described in WO2000/042072.

In a non-limiting embodiment of the present invention, examples of apreferred Fc region include Fc regions having one or more of thefollowing alterations as indicated by EU numbering in an aforementionedFc region:

Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, orTrp for the amino acid at position 234;Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, orArg for the amino acid at position 235;Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, or Tyr for the amino acidat position 236;Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val,Tyr, or Arg for the amino acid at position 237;Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, or Arg for the aminoacid at position 238;Gln, His, Lys, Phe, Pro, Trp, Tyr, or Arg for the amino acid at position239;Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp,Tyr, or Val for the amino acid at position 265;Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, orTyr for the amino acid at position 266;Arg, His, Lys, Phe, Pro, Trp, or Tyr for the amino acid at position 267;Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val for the amino acid at position 269;Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val for the amino acid at position 270;Arg, His, Phe, Ser, Thr, Trp, or Tyr for the amino acid at position 271;Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid atposition 295;Arg, Gly, Lys, or Pro for the amino acid at position 296;Ala for the amino acid at position 297;Arg, Gly, Lys, Pro, Trp, or Tyr for the amino acid at position 298;Arg, Lys, or Pro for the amino acid at position 300;Lys or Pro for the amino acid at position 324;Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, or Val for theamino acid at position 325;Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Valfor the amino acid at position 327;Arg, Asn, Gly, His, Lys, or Pro for the amino acid at position 328;Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp,Tyr, Val, or Arg for the amino acid at position 329;Pro or Ser for the amino acid at position 330;Arg, Gly, or Lys for the amino acid at position 331; orArg, Lys, or Pro for the amino acid at position 332.

Embodiment 2 An Antigen-Binding Molecule Containing an Fc Region HavingFcRn-Binding Activity Under a Neutral pH Range Condition and WhoseBinding Activity Toward an Inhibitory FcγR is Higher than the BindingActivity Toward an Activating Fcγ Receptor

By binding to two molecules of FcRn and one molecule of inhibitory FcγR,the antigen-binding molecule of Embodiment 2 can form a complexcomprising these four molecules. However, since a single antigen-bindingmolecule can bind with only one molecule of FcγR, the singleantigen-binding molecule in a state bound to an inhibitory FcγR cannotbind to other activating FcγRs. Furthermore, it has been reported thatan antigen-binding molecule that is incorporated into cells in a statebound to an inhibitory FcγR is recycled onto the cell membrane, and thusescapes from degradation inside the cells (Immunity (2005) 23, 503-514).More specifically, it is considered that antigen-binding moleculeshaving selective binding activity toward an inhibitory FcγR cannot formheterocomplexes containing an activating FcγR and two molecules of FcRn,which cause an immune response.

Preferred activating Fcγ receptors include FcγRI (CD64) which includesFcγRIa, FcγRIb, and FcγRIc; FcγRIIa (including allotypes R131 and H131);and FcγRIII (CD16) which includes isoforms FcγRIIIa (including allotypesV158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 andFcγRIIIb-NA2). Meanwhile, examples of preferred inhibitory Fcγ receptorsinclude FcγRIIb (including FcγRIIb-1 and FcγRIIb-2).

Herein, “a binding activity toward an inhibitory FcγR is higher than thebinding activity toward an activating Fcγ receptor” means that thebinding activity of the Fc region variant toward FcγRIIb is higher thanthe binding activity toward any of the human Fcγ receptors, FcγRI,FcγRIIa, FcγRIIIa, and/or FcγRIIIb. For example, it means that based onan above-described analytical method, the binding activity towardFcγRIIb of the antigen-binding molecule containing an Fc region variantas compared with the binding activity toward any of the human Fcγreceptors, FcγRI, FcγRIIa, FcγRIIIa, and/or FcγRIIIb is 105% or more,preferably 110% or more, 120% or more, 130% or more, 140% or more, andparticularly preferably 150% or more, 160% or more, 170% or more, 180%or more, 190% or more, 200% or more, 250% or more, 300% or more, 350% ormore, 400% or more, 450% or more, 500% or more, 750% or more, 10 timesor more, 20 times or more, 30 times or more, 40 times or more, 50 timesor more.

Most preferably, the binding activity toward FcγRIIb is higher than eachof the binding activities toward FcγRIa, FcγRIIa (including allotypesR131 and H131), and FcγRIIIa (including allotypes V158 and F158). FcγRIashows a markedly high affinity toward native IgG1; thus, the binding isthought to be saturated in vivo due to the presence of a large amount ofendogenous IgG1. For this reason, inhibition of complex formation may bepossible even if the binding activity toward FcγRIIb is greater than thebinding activities toward FcγRIIa and FcγRIIIa, and lower than thebinding activity toward FcγRIa.

As a control antigen-binding molecule containing an Fc region,antigen-binding molecules having an Fc region of a monoclonal IgGantibody may be appropriately used. The structures of such Fc regionsare shown in SEQ ID NO: 5 (A is added to the N terminus of RefSeqAccession No. AAC82527.1), SEQ ID NO: 6 (A is added to the N terminus ofRefSeq Accession No. AAB59393.1), SEQ ID NO: 7 (RefSeq Accession No.CAA27268.1), and SEQ ID NO: 8 (A is added to the N terminus of RefSeqAccession No. AAB59394.1). Further, when an antigen-binding moleculecontaining an Fc region of a particular antibody isotype is used as thetest substance, effect on the binding activity of the Fcregion-containing antigen-binding molecule toward an Fcγ receptor istested by using an antigen-binding molecule having the Fc region of amonoclonal IgG antibody of a particular isotype as a control. In thisway, antigen-binding molecules containing an Fc region whose bindingactivity toward the Fcγ receptor was demonstrated to be high areappropriately selected.

In a non-limiting embodiment of the present invention, preferredexamples of Fc regions having a selective binding activity toward aninhibitory FcγR include Fc regions in which among the amino acids of anabove-described Fc region, the amino acid at 238 or 328 as indicated byEU numbering is altered to an amino acid different from that of thenative Fc region. Furthermore, as an Fc region having a selectivebinding activity toward an inhibitory Fcγ receptor, the Fc regions oralterations described in US 2009/0136485 can be appropriately selected.

In a non-limiting embodiment of the present invention, a preferredexample is an Fc region having one or more of the following alterationsas indicated by EU numbering in an aforementioned Fc region: the aminoacid at position 238 is Asp; or the amino acid at position 328 is Glu.

In still another non-limiting embodiment of the present invention,examples of a preferred Fc region include Fc regions having asubstitution of Pro at position 238 according to EU numbering with Aspand having one or more of the alterations:

alteration of the amino acid at position 237 according to EU numberingto Trp, the amino acid at position 237 according to EU numbering is Phe,the amino acid at position 267 according to EU numbering is Val, theamino acid at position 267 according to EU numbering is Gln, the aminoacid at position 268 according to EU numbering is Asn, the amino acid atposition 271 according to EU numbering is Gly, the amino acid atposition 326 according to EU numbering is Leu, the amino acid atposition 326 according to EU numbering is Gln, the amino acid atposition 326 according to EU numbering is Glu, the amino acid atposition 326 according to EU numbering is Met, the amino acid atposition 239 according to EU numbering is Asp, the amino acid atposition 267 according to EU numbering is Ala, the amino acid atposition 234 according to EU numbering is Trp, the amino acid atposition 234 according to EU numbering is Tyr, the amino acid atposition 237 according to EU numbering is Ala, the amino acid atposition 237 according to EU numbering is Asp, the amino acid atposition 237 according to EU numbering is Glu, the amino acid atposition 237 according to EU numbering is Leu, the amino acid atposition 237 according to EU numbering is Met, the amino acid atposition 237 according to EU numbering is Tyr, the amino acid atposition 330 according to EU numbering is Lys, the amino acid atposition 330 according to EU numbering is Arg, the amino acid atposition 233 according to EU numbering is Asp, the amino acid atposition 268 according to EU numbering is Asp, the amino acid atposition 268 according to EU numbering is Glu, the amino acid atposition 326 according to EU numbering is Asp, the amino acid atposition 326 according to EU numbering is Ser, the amino acid atposition 326 according to EU numbering is Thr, the amino acid atposition 323 according to EU numbering is Ile, the amino acid atposition 323 according to EU numbering is Leu, the amino acid atposition 323 according to EU numbering is Met, the amino acid atposition 296 according to EU numbering is Asp, the amino acid atposition 326 according to EU numbering is Ala, the amino acid atposition 326 according to EU numbering is Asn, and the amino acid atposition 330 according to EU numbering is Met.

Embodiment 3 An Antigen-Binding Molecule Containing an Fc Region, inwhich One of the Two Polypeptides Constituting the Fc Region has anFcRn-Binding Activity Under a Neutral pH Range Condition and the OtherPolypeptide does not have FcRn-Binding Activity Under a Neutral pH RangeCondition

By binding to one molecule of FcRn and one molecule of FcγR, theantigen-binding molecule of Embodiment 3 can form a trimeric complex;however, it does not form any heterocomplex comprising four moleculesincluding two molecules of FcRn and one molecule of FcγR. As an Fcregion in which one of the two polypeptides constituting the Fc regionhas an FcRn-binding activity under a neutral pH range condition and theother does not have any FcRn-binding activity under a neutral pH rangecondition contained in the antigen-binding molecule of Embodiment 3, Fcregions derived from bispecific antibodies may be suitably used.Bispecific antibodies are two types of antibodies having specificitiestoward different antigens. Bispecific antibodies of an IgG type can besecreted from hybrid hybridomas (quadromas) resulting from fusion of twotypes of hybridomas producing IgG antibodies (Milstein et al. (Nature(1983) 305, 537-540).

When an antigen-binding molecule of Embodiment 3 described above isproduced by using recombination techniques such as those described inthe section “Antibodies” above, one can use a method in which genesencoding the polypeptides that constitute the two types of Fc regions ofinterest are transfected into cells to co-express them. However, theproduced Fc regions will be a mixture in which the following will existat a molecular ratio of 2:1:1: an Fc region in which one of the twopolypeptides constituting the Fc region has an FcRn-binding activityunder a neutral pH range condition and the other polypeptide does nothave any FcRn-binding activity under a neutral pH range condition; an Fcregion in which the two polypeptides constituting the Fc region bothhave an FcRn-binding activity under a neutral pH range condition; and anFc region in which both of the two polypeptides constituting the Fcregion do not have FcRn-binding activity under a neutral pH rangecondition. It is difficult to purify antigen-binding moleculescontaining the desired combination of Fc regions from the three types ofIgGs.

When producing the antigen-binding molecules of Embodiment 3 using suchrecombination techniques, antigen-binding molecules comprising aheteromeric combination of Fc regions can be preferentially secreted byadding appropriate amino acid substitutions to the CH3 domainsconstituting the Fc regions. Specifically, this method is conducted bysubstituting an amino acid having a larger side chain (knob (which means“bulge”)) for an amino acid in the CH3 domain of one of the heavychains, and substituting an amino acid having a smaller side chain (hole(which means “void”)) for an amino acid in the CH3 domain of the otherheavy chain so that the knob is arranged in the hole. This promotesheteromeric H chain formation and simultaneously inhibits homomeric Hchain formation (WO 1996027011; Ridgway et al., (Protein Engineering(1996) 9, 617-621); Merchant et al., (Nature Biotechnology (1998) 16,677-681)).

Furthermore, there are also known techniques for producing a bispecificantibody by applying methods for controlling polypeptide association orassociation of polypeptide-formed heteromeric multimers to theassociation between two polypeptides that constitute an Fc region.Specifically, methods for controlling polypeptide association may beemployed to produce a bispecific antibody (WO 2006/106905), in whichamino acid residues forming the interface between two polypeptides thatconstitute the Fc region are altered to inhibit the association betweenFc regions having the same sequence, and to allow the formation ofpolypeptide complexes formed by two Fc regions of different sequences.Specifically, the methods in the above-described section on bispecificantibodies and methods for producing them can be used as a non-limitingembodiment for preparing the antigen-binding molecule of Embodiment 3 ofthe present invention.

These antigen-binding molecules of Embodiments 1 to 3 are all expectedto be able to reduce immunogenicity and improve plasma retention ascompared to antigen-binding molecules capable of forming tetramericcomplexes.

Methods for Producing Antigen-Binding Domains

The present invention provides methods for producing antigen-bindingdomains whose antigen-binding activity in the presence of a targettissue-specific compound is higher than the antigen-binding activity inthe absence of the compound.

More specifically, the present invention provides a method for producingan antigen-binding domain, which comprises steps (a) to (e) below:

-   (a) determining the antigen-binding activitiy of an antigen-binding    domain in the absence of a target tissue-specific compound;-   (b) determining the antigen-binding activity of an antigen-binding    domain in the presence of the target tissue-specific compound;-   (c) selecting an antigen-binding domain whose antigen-binding    activity in the absence of a target tissue-specific compound is    lower than in the presence of the compound;-   (d) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (c)    is operably linked; and-   (e) collecting an antigen-binding domain from a culture medium of    the cells cultured in (d).

The present invention also provides a method for producing anantigen-binding domain, which comprises steps (a) to (e) below:

-   (a) determining the antigen-binding activity of an antigen-binding    domain in the presence of a low concentration of a target    tissue-specific compound;-   (b) determining the antigen-binding activity of an antigen-binding    domain in the presence of a high concentration of the target    tissue-specific compound;-   (c) selecting an antigen-binding domain whose antigen-binding    activity in the presence of a low concentration of the target    tissue-specific compound is lower than in the presence of a high    concentration of the compound;-   (d) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (c)    is operably linked; and-   (e) collecting an antigen-binding domain from a culture medium of    the cells cultured in (d).

Furthermore, the present invention provides a method for producing anantigen-binding domain, which comprises steps (a) to (e) below:

-   (a) contacting antigen-binding domains or a library thereof with an    antigen in the presence of a target tissue-specific compound;-   (b) placing the antigen-binding domains that bound to the antigen in    said step (a) in the absence of the compound;-   (c) isolating an antigen-binding domain that was dissociated in said    step (b);-   (d) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (c)    is operably linked; and-   (e) collecting an antigen-binding domain from a culture medium of    the cells cultured in (d).

In addition, the present invention provides a method for producing anantigen-binding domain, which comprises steps (a) to (e) below:

-   (a) contacting antigen-binding domains or a library thereof to an    antigen in the presence of a high concentration of a target    tissue-specific compound;-   (b) placing the antigen-binding domains that bind to the antigen in    said step (a) in the presence of a low concentration of the    compound;-   (c) isolating an antigen-binding domain that dissociates in said    step (b);-   (d) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (c)    is operably linked; and-   (e) collecting an antigen-binding domain from a culture medium of    the cells cultured in (d).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps of (a) to (f) below:

-   (a) contacting a library of antigen-binding domains with an antigen    in the absence of a target tissue-specific compound;-   (b) selecting antigen-binding domains that do not bind to the    antigen in said step (a);-   (c) allowing the antigen-binding domains selected in said step (b)    to bind to the antigen in the presence of the compound;-   (d) isolating an antigen-binding domain that bind to the antigen in    said step (c);-   (e) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (d)    is operably linked; and-   (f) collecting an antigen-binding domain from a culture medium of    the cells cultured in (e).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (f) below:

-   (a) contacting a library of antigen-binding domains with an antigen    in the presence of a low concentration of a target tissue-specific    compound;-   (b) selecting antigen-binding domains that do not bind to the    antigen in said step (a);-   (c) allowing the antigen-binding domains selected in said step (b)    to bind to the antigen in the presence of a high concentration of    the compound;-   (d) isolating an antigen-binding domain that bind to the antigen in    said step (c);-   (e) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (d)    is operably linked; and-   (f) collecting an antigen-binding domain from a culture medium of    the cells cultured in (e).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (e) below:

-   (a) contacting a library of antigen-binding domains with an    antigen-immobilized column in the presence of a target    tissue-specific compound;-   (b) eluting antigen-binding domains that bind to the column in said    step (a) from the column in the absence of the compound;-   (c) isolating the antigen-binding domain eluted in said step (b);-   (d) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (c)    is operably linked; and-   (e) collecting an antigen-binding domain from a culture medium of    the cells cultured in (d).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (e) below:

-   (a) contacting a library of antigen-binding domains with an    antigen-immobilized column in the presence of a high concentration    of a target tissue-specific compound;-   (b) eluting antigen-binding domains that bind to the column in said    step (a) from the column in the presence of a low concentration of    the compound;-   (c) isolating an antigen-binding domain eluted in said step (b);-   (d) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (c)    is operably linked; and-   (e) collecting an antigen-binding domain from a culture medium of    the cells cultured in (d).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (f) below:

-   (a) allowing a library of antigen-binding domains to pass through an    antigen-immobilized column in the absence of a target    tissue-specific compound;-   (b) collecting antigen-binding domains that are eluted without    binding to the column in step (a);-   (c) allowing the antigen-binding domains collected in step (b) to    bind to the antigen in the presence of the compound;-   (d) isolating an antigen-binding domain that bind to the antigen in    step (c);-   (e) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (d)    is operably linked; and-   (f) collecting an antigen-binding domain from a culture medium of    the cells cultured in (e).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (f) below:

-   (a) allowing a library of antigen-binding domains to pass through an    antigen-immobilized column in the presence of a low concentration of    a target tissue-specific compound;-   (b) collecting antigen-binding domains that are eluted without    binding to the column in said step (a);-   (c) allowing the antigen-binding domains collected in said step (b)    to bind to the antigen in the presence of a high concentration of    the compound;-   (d) isolating an antigen-binding domain that binds to the antigen in    said step (c);-   (e) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (d)    is operably linked; and-   (f) collecting an antigen-binding domain from a culture medium of    the cells cultured in (e).

Furthermore, the present invention provides a method for producing anantigen-binding domain, which comprises steps (a) to (f) below:

-   (a) contacting an antigen with a library of antigen-binding domains    in the presence of a target tissue-specific compound;-   (b) obtaining antigen-binding domains that bind to the antigen in    step (a);-   (c) placing the antigen-binding domain obtained in step (b) in the    absence of the compound;-   (d) isolating an antigen-binding domain whose antigen-binding    activity in step (c) is weaker than the reference selected in step    (b);-   (e) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (d)    is operably linked; and-   (f) collecting an antigen-binding domain from a culture medium of    the cells cultured in (e).

The present invention provides a method for producing an antigen-bindingdomain, which comprises steps (a) to (f) below:

-   (a) contacting an antigen with a library of antigen-binding domains    in the presence of a high concentration of a target tissue-specific    compound;-   (b) obtaining antigen-binding domains that bind to the antigen in    step (a);-   (c) placing the antigen-binding domains obtained in step (b) in the    presence of a low concentration of the compound;-   (d) isolating an antigen-binding domain whose antigen-binding    activity in step (c) is weaker than the reference selected in step    (b);-   (e) culturing cells transfected with a vector to which a    polynucleotide encoding the antigen-binding domain selected in (d)    is operably linked; and-   (f) collecting an antigen-binding domain from a culture medium of    the cells cultured in (e).

The terms “cells”, “cell line”, and “cell culture” are used synonymouslyherein, and such naming may include all progenies of the cells or cellline. This way, for example, the terms “transformant” and “transformedcells” include cultures and primary target cells derived from themregardless of the number of passages. Furthermore, it is understood thatdue to intentional or accidental mutations, the DNA content is notalways exactly the same in all progenies. Progenies of mutants havingsubstantially the same function or biological activity such as thosescreened for in the initially transformed cells may also be included.When the description is intended to refer to a different naming, thatintention may become obvious from the context of the description. Cellsthat are appropriate for use are suitably selected from cells describedin the section “Antibodies” above.

When referring to the expression of a coding sequence, the term “controlsequences” refers to DNA nucleotide sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotesinclude, for example, a promoter, optionally an operator sequence, aribosome binding site, and possibly, other as yet poorly understoodsequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers for the expression of a codingsequence.

For a nucleic acid, the term “operably linked” means that the nucleicacid is placed into a functional relationship with another nucleic acidsequence. For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as aprecursor protein that participates in the secretion of the polypeptide.A promoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. A ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading frame. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at suitablerestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice. Furthermore, linked nucleic acids may be producedby the above-mentioned overlap extension PCR technique.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo nucleic acid fragments. For ligation of the two fragments, the endsof the fragments must be compatible with each other. In some cases, theends will be directly compatible after endonuclease digestion. However,it may be necessary first to convert the staggered ends commonlyproduced after endonuclease digestion to blunt ends to make themcompatible for ligation. For blunting the ends, the DNA is treated in asuitable buffer for at least 15 minutes at 15° C. with about 10 units ofthe Klenow fragment of DNA polymerase I or T4 DNA polymerase in thepresence of the four deoxyribonucleotide triphosphates. The DNA is thenpurified by phenol-chloroform extraction and ethanol precipitation, orby silica purification. The DNA fragments that are to be ligatedtogether are put in solution in equimolar amounts. The solution willcontain ATP, ligase buffer, and a ligase such as T4 DNA ligase at about10 units per 0.5 μg of DNA. If the DNA is to be ligated into a vector,the vector is first linearized by digestion with the appropriaterestriction endonuclease(s). The linearized fragment is then treatedwith bacterial alkaline phosphatase or calf intestinal phosphatase toprevent self-ligation of the fragment during the ligation step.

In the production methods of the present invention, an antigen-bindingdomain which has a higher antigen-binding activity in the presence of atarget tissue-specific compound than in its absence, which has beenselected by the method described in the above section “Antigen-bindingdomain dependent on a compound specific to a target tissue” is isolated.For example, when an antigen-binding domain isolated in this manner hasbeen selected from a library, the polynucleotide encoding theantigen-binding domain is isolated by general gene amplification from avirus such as a phage, as described in the Examples below. Furthermore,when an antigen-binding domain or an antibody isolated in this mannerhas been selected from culture media of cells such as hybridomas, theantibody gene or such can be isolated by general gene amplification fromthe cells as shown in the section “Antibodies” above.

Methods for Producing Antigen-Binding Molecules

The present invention provides methods for producing antigen-bindingmolecules whose antigen-binding activity in the presence of a targettissue-specific compound is higher than the antigen-binding activity inthe absence of the compound.

More specifically, the present invention provides a method for producingantigen-binding molecules, which comprises the steps of:

(a) determining the antigen-binding activity of an antigen-bindingdomain in the absence of a target tissue-specific compound;

(b) determining the antigen-binding activity of the antigen-bindingdomain in the presence of the target tissue-specific compound;

(c) selecting an antigen-binding domain with lower antigen-bindingactivity in the absence of the target tissue-specific compound than inthe presence of the compound;

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting antigen-binding molecules from a culture medium of thecells cultured in (e).

The present invention also provides a method for producing anantigen-binding molecule, which comprises the steps of:

(a) determining the antigen-binding activity of an antigen-bindingdomain in the presence of a low concentration of a targettissue-specific compound;

(b) determining the antigen-binding activity of the antigen-bindingdomain in the presence of a high concentration of the targettissue-specific compound;

(c) selecting an antigen-binding domain with lower antigen-bindingactivity in the presence of a low concentration of the targettissue-specific compound than in the presence of a high concentration ofthe compound;

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting antigen-binding molecules from a culture medium of thecells cultured in (e).

Furthermore, the present invention provides a method for producing anantigen-binding molecule, which comprises the steps of:

(a) contacting antigen-binding domains or a library thereof with anantigen in the presence of a target tissue-specific compound;

(b) placing the antigen-binding domains that bind to the antigen in saidstep (a) in the absence of the compound;

(c) isolating an antigen-binding domain that dissociates in said step(b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting an antigen-binding molecule from a culture medium of thecells cultured in (e).

In addition, the present invention provides a method for producing anantigen-binding molecule, which comprises the steps of:

(a) contacting antigen-binding domains or a library thereof with anantigen in the presence of a high concentration of a targettissue-specific compound;

(b) placing the antigen-binding domains that bind to the antigen in saidstep (a) in the presence of a low concentration of the compound;

(c) isolating an antigen-binding domain that dissociates in said step(b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting antigen-binding molecules from a culture medium of thecells cultured in (e).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with an antigen inthe absence of a target tissue-specific compound;

(b) selecting antigen-binding domains that do not bind to the antigen insaid step (a);

(c) allowing the antigen-binding domains selected in said step (b) tobind to the antigen in the presence of the compound;

(d) isolating an antigen-binding domain that binds to the antigen insaid step (c);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (f).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with an antigen inthe presence of a low concentration of a target tissue-specificcompound;

(b) selecting antigen-binding domains that do not bind to the antigen insaid step (a);

(c) allowing the antigen-binding domains selected in said step (b) tobind to the antigen in the presence of a high concentration of thecompound;

(d) isolating an antigen-binding domain that binds to the antigen insaid step (c);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (f).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with anantigen-immobilized column in the presence of a target tissue-specificcompound;

(b) eluting antigen-binding domains that bind to the column in said step(a) from the column in the absence of the compound;

(c) isolating an antigen-binding domain eluted in said step (b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting antigen-binding molecules from a culture medium of thecells cultured in (e).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with anantigen-immobilized column in the presence of a high concentration of atarget tissue-specific compound;

(b) eluting antigen-binding domains that bind to the column in said step(a) from the column in the presence of a low concentration of thecompound;

(c) isolating an antigen-binding domain eluted in said step (b);

(d) linking a polynucleotide encoding the antigen-binding domainselected in (c) to a polynucleotide encoding a polypeptide containing anFc region;

(e) culturing cells introduced with a vector to which the polynucleotideobtained in (d) is operably linked; and

(f) collecting antigen-binding molecules from a culture medium of thecells cultured in (e).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) allowing a library of antigen-binding domains to pass through anantigen-immobilized column in the absence of a target tissue-specificcompound;

(b) collecting antigen-binding domains that are eluted without bindingto the column in said step (a);

(c) allowing the antigen-binding domains collected in step (b) to bindto the antigen in the presence of the compound;

(d) isolating an antigen-binding domain that binds to the antigen instep (c);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (f).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) allowing a library of antigen-binding domains to pass through anantigen-immobilized column in the presence of a low concentration of atarget tissue-specific compound;

(b) collecting antigen-binding domains that are eluted without bindingto the column in said step (a);

(c) allowing the antigen-binding domains collected in said step (b) tobind to the antigen in the presence of a high concentration of thecompound;

(d) isolating an antigen-binding domain that binds to the antigen insaid step (c);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (f).

Furthermore, the present invention provides a method for producing anantigen-binding molecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with an antigen inthe presence of a target tissue-specific compound;

(b) obtaining antigen-binding domains that bind to the antigen in saidstep (a);

(c) placing the antigen-binding domains obtained in said step (b) in theabsence of the compound;

(d) isolating an antigen-binding domain whose antigen-binding activityin said step (c) is weaker than the reference selected in step (b);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (f).

The present invention provides a method for producing an antigen-bindingmolecule, which comprises the steps of:

(a) contacting a library of antigen-binding domains with an antigen inthe presence of a high concentration of a target tissue-specificcompound;

(b) obtaining antigen-binding domains that bind to the antigen in saidstep (a);

(c) placing the antigen-binding domains obtained in step (b) in thepresence of a low concentration of the compound;

(d) isolating an antigen-binding domain whose antigen-binding activityin step (c) is weaker than the reference selected in step (b);

(e) linking a polynucleotide encoding the antigen-binding domainselected in (d) to a polynucleotide encoding a polypeptide containing anFc region;

(f) culturing cells introduced with a vector to which the polynucleotideobtained in (e) is operably linked; and

(g) collecting antigen-binding molecules from a culture medium of thecells cultured in (f).

A non-limiting embodiment of the Fc region whose polynucleotide sequenceis linked to a polynucleotide encoding an antigen-binding domain is, forexample, the Fc region contained in the constant region of a human IgG1(SEQ ID NO: 5), IgG2 (SEQ ID NO: 6), IgG3 (SEQ ID NO: 7), or IgG4 (SEQID NO: 8) antibody. An Fc region is a portion of the heavy chainconstant region of an antibody, starting from the N terminal end of thehinge region, which corresponds to the papain cleavage site at an aminoacid around position 216 according to EU numbering, and contains thehinge, CH2, and CH3 domains. The Fc region may be obtained from humanIgG1, but it is not limited to any particular subclass of IgG.

A non-limiting embodiment of the Fc region whose polynucleotide sequenceis linked to a polynucleotide encoding an antigen-binding domainincludes, for example, Fc regions whose Fcγ receptor-binding activity ishigher than the Fcγ receptor-binding activity of the Fc region of anative human IgG. Examples of such Fc regions include Fc regions inwhich at least one or more amino acids selected from the groupconsisting of amino acids at positions 221, 222, 223, 224, 225, 227,228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243,244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278,279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315,317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392,396, 421, 427, 428, 429, 434, 436, and 440, according to EU numbering,are different from the corresponding amino acid residues according to EUnumbering in the Fc region contained in the antibody constant region ofSEQ ID NO: 5, 6, 7, or 8.

Furthermore, a non-limiting embodiment of the above-mentioned Fc regionincludes, for example, Fc regions comprising at least one or more aminoacid alterations selected from the group consisting of:

alteration of the amino acid at position 221 to Lys or Tyr;alteration of the amino acid at position 222 to Phe, Trp, Glu, or Tyr;alteration of the amino acid at position 223 to Phe, Trp, Glu, or Lys;alteration of the amino acid at position 224 to Phe, Trp, Glu, or Tyr;alteration of the amino acid at position 225 to Glu, Lys, or Trp;alteration of the amino acid at position 227 to Glu, Gly, Lys, or Tyr;alteration of the amino acid at position 228 to Glu, Gly, Lys, or Tyr;alteration of the amino acid at position 230 to Ala, Glu, Gly, or Tyr;alteration of the amino acid at position 231 to Glu, Gly, Lys, Pro, orTyr;alteration of the amino acid at position 232 to Glu, Gly, Lys, or Tyr;alteration of the amino acid at position 233 to Ala, Asp, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 234 to Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 235 to Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 236 to Ala, Asp, Glu, Phe, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 237 to Asp, Glu, Phe, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 238 to Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 239 to Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 240 to Ala, Ile, Met, or Thr;alteration of the amino acid at position 241 to Asp, Glu, Leu, Arg, Trp,or Tyr;alteration of the amino acid at position 243 to Leu, Glu, Leu, Gln, Arg,Trp, or Tyr;alteration of the amino acid at position 244 to His;alteration of the amino acid at position 245 to Ala;alteration of the amino acid at position 246 to Asp, Glu, His, or Tyr;alteration of the amino acid at position 247 to Ala, Phe, Gly, His, Ile,Leu, Met, Thr, Val, or Tyr;alteration of the amino acid at position 249 to Glu, His, Gln, or Tyr;alteration of the amino acid at position 250 to Glu, or Gln;alteration of the amino acid at position 251 to Phe;alteration of the amino acid at position 254 to Phe, Met, or Tyr;alteration of the amino acid at position 255 to Glu, Leu, or Tyr;alteration of the amino acid at position 256 to Ala, Met, or Pro;alteration of the amino acid at position 258 to Asp, Glu, His, Ser, orTyr;alteration of the amino acid at position 260 to Asp, Glu, His, or Tyr;alteration of the amino acid at position 262 to Ala, Glu, Phe, Ile, orThr;alteration of the amino acid at position 263 to Ala, Ile, Met, or Thr;alteration of the amino acid at position 264 to Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;alteration of the amino acid at position 265 to Ala, Leu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 266 to Ala, Ile, Met, or Thr;alteration of the amino acid at position 267 to Asp, Glu, Phe, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 268 to Asp, Glu, Phe, Gly, Ile,Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp;alteration of the amino acid at position 269 to Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 270 to Glu, Phe, Gly, His, Ile,Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;alteration of the amino acid at position 271 to Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 272 to Asp, Phe, Gly, His, Ile,Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 273 to Phe or Ile;alteration of the amino acid at position 274 to Asp, Glu, Phe, Gly, His,Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 275 to Leu or Trp;alteration of the amino acid at position 276 to Asp, Glu, Phe, Gly, His,Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 278 to Asp, Glu, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp;alteration of the amino acid at position 279 to Ala;alteration of the amino acid at position 280 to Ala, Gly, His, Lys, Leu,Pro, Gln, Trp, or Tyr;alteration of the amino acid at position 281 to Asp, Lys, Pro, or Tyr;alteration of the amino acid at position 282 to Glu, Gly, Lys, Pro, orTyr;alteration of the amino acid at position 283 to Ala, Gly, His, Ile, Lys,Leu, Met, Pro, Arg, or Tyr;alteration of the amino acid at position 284 to Asp, Glu, Leu, Asn, Thr,or Tyr;alteration of the amino acid at position 285 to Asp, Glu, Lys, Gln, Trp,or Tyr;alteration of the amino acid at position 286 to Glu, Gly, Pro, or Tyr;alteration of the amino acid at position 288 to Asn, Asp, Glu, or Tyr;alteration of the amino acid at position 290 to Asp, Gly, His, Leu, Asn,Ser, Thr, Trp, or Tyr;alteration of the amino acid at position 291 to Asp, Glu, Gly, His, Ile,Gln, or Thr;alteration of the amino acid at position 292 to Ala, Asp, Glu, Pro, Thr,or Tyr;alteration of the amino acid at position 293 to Phe, Gly, His, Ile, Leu,Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 294 to Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 295 to Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 296 to Ala, Asp, Glu, Gly, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, or Val;alteration of the amino acid at position 297 to Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 298 to Ala, Asp, Glu, Phe, His,Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 299 to Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr;alteration of the amino acid at position 300 to Ala, Asp, Glu, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp;alteration of the amino acid at position 301 to Asp, Glu, His, or Tyr;alteration of the amino acid at position 302 to Ile;alteration of the amino acid at position 303 to Asp, Gly, or Tyr;alteration of the amino acid at position 304 to Asp, His, Leu, Asn, orThr;alteration of the amino acid at position 305 to Glu, Ile, Thr, or Tyr;alteration of the amino acid at position 311 to Ala, Asp, Asn, Thr, Val,or Tyr;alteration of the amino acid at position 313 to Phe;alteration of the amino acid at position 315 to Leu;alteration of the amino acid at position 317 to Glu or Gln;alteration of the amino acid at position 318 to His, Leu, Asn, Pro, Gln,Arg, Thr, Val, or Tyr;alteration of the amino acid at position 320 to Asp, Phe, Gly, His, Ile,Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 322 to Ala, Asp, Phe, Gly, His,Ile, Pro, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 323 to Ile;alteration of the amino acid at position 324 to Asp, Phe, Gly, His, Ile,Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 325 to Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 326 to Ala, Asp, Glu, Gly, Ile,Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 327 to Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 328 to Ala, Asp, Glu, Phe, Gly,His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 329 to Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 330 to Cys, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 331 to Asp, Phe, His, Ile, Leu,Met, Gln, Arg, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 332 to Ala, Asp, Glu, Phe, Gly,His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid at position 333 to Ala, Asp, Glu, Phe, Gly,His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyr;alteration of the amino acid at position 334 to Ala, Glu, Phe, Ile, Leu,Pro, or Thr;alteration of the amino acid at position 335 to Asp, Phe, Gly, His, Ile,Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr;alteration of the amino acid at position 336 to Glu, Lys, or Tyr;alteration of the amino acid at position 337 to Glu, His, or Asn;alteration of the amino acid at position 339 to Asp, Phe, Gly, Ile, Lys,Met, Asn, Gln, Arg, Ser, or Thr;alteration of the amino acid at position 376 to Ala or Val;alteration of the amino acid at position 377 to Gly or Lys;alteration of the amino acid at position 378 to Asp;alteration of the amino acid at position 379 to Asn;alteration of the amino acid at position 380 to Ala, Asn, or Ser;alteration of the amino acid at position 382 to Ala or Ile;alteration of the amino acid at position 385 to Glu;alteration of the amino acid at position 392 to Thr;alteration of the amino acid at position 396 to Leu;alteration of the amino acid at position 421 to Lys;alteration of the amino acid at position 427 to Asn;alteration of the amino acid at position 428 to Phe or Leu;alteration of the amino acid at position 429 to Met;alteration of the amino acid at position 434 to Trp;alteration of the amino acid at position 436 to Ile; andalteration of the amino acid at position 440 to Gly, His, Ile, Leu, orTyr;according to EU numbering, in the amino acid residues of the Fc regioncontained in the antibody constant region of SEQ ID NO: 5, 6, 7, or 8.The number of altered amino acids is not particularly limited; an aminoacid at only one site may be altered, or amino acids at two or moresites may be altered. Combinations of amino acid alterations at two ormore sites include, for example, those described in Table 1 (Table 1-1to Table 1-3).

A non-limiting embodiment of the Fc region whose polynucleotide sequenceis linked to a polynucleotide encoding an antigen-binding domain is, forexample, an Fc region having binding activity toward an inhibitory Fcγreceptor that is higher than the binding activity toward an activatingFcγ receptor. Specifically, a non-limiting embodiment of such Fc regionsis an Fc region whose binding activity to FcγRIIb is higher than thebinding activity toward any of the human Fcγ receptors FcγRIa, FcγRIIa,FcγRIIIa, and/or FcγRIIIb.

A non-limiting embodiment of the above-mentioned Fc region preferablyincludes, for example, an Fc region in which the amino acid at 238 or328 according to EU numbering in the Fc region contained in the antibodyconstant region of SEQ ID NO: 5, 6, 7, or 8, is altered to an amino aciddifferent from that of the native Fc region. A preferred example of suchFc regions is an Fc region having one or more of the followingalterations: alteration of the amino acid at position 238 to Asp, andalteration of the amino acid at position 328 to Glu, according to EUnumbering, in the aforementioned Fc region.

In still another non-limiting embodiment of the above-mentioned Fcregion, preferred examples include Fc regions having one or more of thealterations exemplified in PCT/JP2012/054624: substitution of Pro atposition 238 according to EU numbering with Asp, alteration of the aminoacid at position 237 according to EU numbering to Trp, alteration of theamino acid at position 237 according to EU numbering to Phe, alterationof the amino acid at position 267 according to EU numbering to Val,alteration of the amino acid at position 267 according to EU numberingto Gln, alteration of the amino acid at position 268 according to EUnumbering to Asn, alteration of the amino acid at position 271 accordingto EU numbering to Gly, alteration of the amino acid at position 326according to EU numbering to Leu, alteration of the amino acid atposition 326 according to EU numbering to Gln, alteration of the aminoacid at position 326 according to EU numbering to Glu, alteration of theamino acid at position 326 according to EU numbering to Met, alterationof the amino acid at position 239 according to EU numbering to Asp,alteration of the amino acid at position 267 according to EU numberingto Ala, alteration of the amino acid at position 234 according to EUnumbering to Trp, alteration of the amino acid at position 234 accordingto EU numbering to Tyr, alteration of the amino acid at position 237according to EU numbering to Ala, alteration of the amino acid atposition 237 according to EU numbering to Asp, alteration of the aminoacid at position 237 according to EU numbering to Glu, alteration of theamino acid at position 237 according to EU numbering to Leu, alterationof the amino acid at position 237 according to EU numbering to Met,alteration of the amino acid at position 237 according to EU numberingto Tyr, alteration of the amino acid at position 330 according to EUnumbering to Lys, alteration of the amino acid at position 330 accordingto EU numbering to Arg, alteration of the amino acid at position 233according to EU numbering to Asp, alteration of the amino acid atposition 268 according to EU numbering to Asp, alteration of the aminoacid at position 268 according to EU numbering to Glu, alteration of theamino acid at position 326 according to EU numbering to Asp, alterationof the amino acid at position 326 according to EU numbering to Ser,alteration of the amino acid at position 326 according to EU numberingto Thr, alteration of the amino acid at position 323 according to EUnumbering to Ile, alteration of the amino acid at position 323 accordingto EU numbering to Leu, alteration of the amino acid at position 323according to EU numbering to Met, alteration of the amino acid atposition 296 according to EU numbering to Asp, alteration of the aminoacid at position 326 according to EU numbering to Ala, alteration of theamino acid at position 326 according to EU numbering to Asn, andalteration of the amino acid at position 330 according to EU numberingto Met.

A non-limiting embodiment of the Fc region whose polynucleotide sequenceis linked to a polynucleotide encoding an antigen-binding domainincludes Fc regions having binding activity to FcRn in the acidic pHrange. Amino acids that can undergo such alteration include, forexample, amino acids at positions 252, 254, 256, 309, 311, 315, 433,and/or 434 according to EU numbering, and amino acids at positions 253,310, 435, and/or 426 which are combined with the above amino acids, asdescribed in WO 1997/034631. Preferred examples include amino acids atpositions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305,307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386,388, 400, 413, 415, 424, 433, 434, 435, 436, 439, and/or 447 (EUnumbering) as described in WO 2000/042072. Similarly, preferred examplesof amino acids that can undergo such alteration include amino acids atpositions 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387,389, 428, 433, 434, and/or 436 according to EU numbering as described inWO 2002/060919. Furthermore, amino acids that can undergo suchalteration are, for example, amino acids at positions 250, 314, and 428according to EU numbering as described in WO2004/092219. In addition,preferred examples of amino acids that can undergo such alterationinclude amino acids at positions 238, 244, 245, 249, 252, 256, 257, 258,260, 262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 311, 312, 316,317, 318, 332, 339, 341, 343, 375, 376, 377, 378, 380, 382, 423, 427,430, 431, 434, 436, 438, 440, and/or 442 as described in WO 2006/020114.Furthermore, preferred examples of amino acids that can undergo suchalteration include amino acids at positions 251, 252, 307, 308, 378,428, 430, 434, and/or 436 according to EU numbering as described in WO2010/045193.

A non-limiting embodiment of the above-mentioned Fc region includes, forexample, Fc regions having at least one or more amino acid alterationsselected from the group consisting of:

alteration of the amino acid of position 251 to Arg or Leu;alteration of the amino acid of position 252 to Phe, Ser, Thr, or Tyr;alteration of the amino acid of position 254 to Ser or Thr;alteration of the amino acid of position 255 to Arg, Gly, Ile, or Leu;alteration of the amino acid of position 256 to Ala, Arg, Asn, Asp, Gln,Glu, or Thr;alteration of the amino acid of position 308 to Ile or Thr;alteration of the amino acid of position 309 to Pro;alteration of the amino acid of position 311 to Glu, Leu, or Ser;alteration of the amino acid of position 312 to Ala or Asp;alteration of the amino acid of position 314 to Ala or Leu;alteration of the amino acid of position 385 to Ala, Arg, Asp, Gly, His,Lys, Ser, or Thr;alteration of the amino acid of position 386 to Arg, Asp, Ile, Lys, Met,Pro, Ser, or Thr;alteration of the amino acid of position 387 to Ala, Arg, His, Pro, Ser,or Thr;alteration of the amino acid of position 389 to Asn, Pro, or Ser;alteration of the amino acid of position 428 to Leu, Met, Phe, Ser, orThr;alteration of the amino acid of position 433 to Arg, Gln, His, Ile, Lys,Pro, or Ser;alteration of the amino acid of position 434 to His, Phe, or Tyr; andalteration of the amino acid of position 436 to Arg, Asn, His, Lys, Met,or Thr,according to EU numbering, in the amino acid residues of the Fc regioncontained in the antibody constant region of SEQ ID NO: 5, 6, 7, or 8.The number of amino acids to be altered is not particularly limited; anamino acid at only one site may be altered or amino acids at two or moresites may be altered.

Another non-limiting embodiment of the Fc region whose binding activityto FcRn in the acidic pH range is stronger than the binding activity ofthe starting Fc region of human IgG1 includes Fc regions in which theamino acid at position 308 is Ile, the amino acid at position 309 isPro, and/or the amino acid at position 311 is Glu, according to EUnumbering, in the amino acid residues of the Fc region contained in theantibody constant region of SEQ ID NO: 5, 6, 7, or 8. Anothernon-limiting embodiment of this Fc region may include Fc regionscontaining Thr for the amino acid of position 308, Pro for the aminoacid of position 309, Leu for the amino acid of position 311, Ala forthe amino acid of position 312, and/or Ala for the amino acid ofposition 314. Furthermore, yet another non-limiting embodiment of thisalteration may include Fc regions containing Ile or Thr for the aminoacid of position 308, Pro for the amino acid of position 309, Glu, Leu,or Ser for the amino acid of position 311, Ala for the amino acid ofposition 312, and/or Ala or Leu for the amino acid of position 314. Adifferent non-limiting embodiment of this alteration may include Fcregions containing Thr for the amino acid of position 308, Pro for theamino acid of position 309, Ser for the amino acid of position 311, Aspfor the amino acid of position 312, and/or Leu for the amino acid ofposition 314.

Another non-limiting embodiment of the Fc region whose binding activityto FcRn in the acidic pH range is stronger than the binding activity ofthe starting Fc region of human IgG1 includes, for example, Fc regionscontaining Leu for the amino acid of position 251, Tyr for the aminoacid of position 252, Ser or Thr for the amino acid of position 254, Argfor the amino acid of position 255, and/or Glu for the amino acid ofposition 256, according to EU numbering, in the amino acid residues ofthe Fc region included in the antibody constant region of SEQ ID NO: 5,6, 7, or 8.

A different non-limiting embodiment of the Fc region whose bindingactivity to FcRn in the acidic pH range is stronger than the bindingactivity of the starting Fc region of human IgG1 includes, for example,Fc regions containing Leu, Met, Phe, Ser, or Thr for the amino acid ofposition 428, Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid ofposition 433, His, Phe, or Tyr for the amino acid of position 434,and/or Arg, Asn, His, Lys, Met, or Thr for the amino acid of position436, according to EU numbering, in the amino acid residues of the Fcregion included in the antibody constant region of SEQ ID NO: 5, 6, 7,or 8. Moreover, another non-limiting embodiment of this alterationincludes Fc regions containing His or Met for the amino acid of position428 and/or His or Met for the amino acid of position 434.

Another different non-limiting embodiment of the Fc region whose bindingactivity to FcRn in the acidic pH range is stronger than the bindingactivity of the starting Fc region of human IgG1 may be, for example,alterations including Arg for the amino acid of position 385, Thr forthe amino acid of position 386, Arg for the amino acid of position 387,and/or Pro for the amino acid of position 389, according to EUnumbering, in the amino acid residues of the Fc region included in theantibody constant region of SEQ ID NO: 5, 6, 7, or 8. Anothernon-limiting embodiment of this alteration include Fc regions containingAsp for the amino acid of position 385, Pro for the amino acid ofposition 386, and/or Ser for the amino acid of position 389.

Another non-limiting embodiment of the Fc region whose binding activityto FcRn in the acidic pH range is stronger than the binding activity ofthe starting Fc region of human IgG1 includes Fc regions containing atleast one or more amino acids selected from the group consisting of

Gln or Glu for the amino acid of position 250; andLeu or Phe for the amino acid of position 428, according to EUnumbering,in the amino acid residues of the Fc region contained in the antibodyconstant region of SEQ ID NO: 5, 6, 7, or 8.

Another non-limiting embodiment of the Fc region whose binding activityto FcRn in the acidic pH range is stronger than the binding activity ofthe starting Fc region of human IgG1 includes, for example, Fc regionscontaining Gln for the amino acid of position 250, and/or Leu or Phe forthe amino acid of position 428, according to EU numbering, in the aminoacid residues of the Fc region contained in the antibody constant regionof SEQ ID NO: 5, 6, 7, or 8. Another non-limiting embodiment of thisalteration may include Glu for the amino acid of position 250, and/orLeu or Phe for the amino acid of position 428.

Another non-limiting embodiment of the Fc region whose binding activityto FcRn in the acidic pH range is stronger than the binding activity ofthe starting Fc region of human IgG1 includes Fc regions containing atleast two or more amino acids selected from the group consisting of:

Asp or Glu for the amino acid of position 251;Tyr for the amino acid of position 252;Gln for the amino acid of position 307;Pro for the amino acid of position 308;Val for the amino acid of position 378;Ala for the amino acid of position 380;Leu for the amino acid of position 428;Ala or Lys for the amino acid of position 430;Ala, His, Ser, or Tyr for the amino acid of position 434; andIle for the amino acid of position 436;according to EU numbering, in the amino acid residues of the Fc regioncontained in the antibody constant region of SEQ ID NO: 5, 6, 7, or 8.

Another non-limiting embodiment of the Fc region whose binding activityto FcRn in the acidic pH range is stronger than the binding activity ofthe starting Fc region of human IgG1 includes, for example, Fc regionscontaining Gln for the amino acid of position 307, and Ala or Ser forthe amino acid of position 434, according to EU numbering, in the aminoacid residues of the Fc region contained in the antibody constant regionof SEQ ID NO: 5, 6, 7, or 8. Another non-limiting embodiment of this Fcregion includes Fc regions containing Pro for the amino acid of position308, and Ala for the amino acid of position 434. Furthermore, anothernon-limiting embodiment of this Fc region includes Fc regions containingTyr for the amino acid of position 252, and Ala for the amino acid ofposition 434. A different non-limiting embodiment of this Fc regionincludes Fc regions containing Val for the amino acid of position 378,and Ala for the amino acid of position 434. Another differentnon-limiting embodiment of this Fc region includes alterations includingLeu for the amino acid of position 428, and Ala for the amino acid ofposition 434. Another different non-limiting embodiment of this Fcregion includes Fc regions containing Ala for the amino acid of position434, and Ile for the amino acid of position 436. Furthermore, anothernon-limiting embodiment of this alteration includes Fc regionscontaining Pro for the amino acid of position 308, and Tyr for the aminoacid of position 434. In addition, another non-limiting embodiment ofthis alteration includes Fc regions containing Gln for the amino acid ofposition 307, and Ile for the amino acid of position 436.

Another non-limiting embodiment of the Fc region whose binding activityto FcRn in the acidic pH range is stronger than the binding activity ofthe starting Fc region of human IgG1 includes Fc regions containing anyone of Gln for the amino acid of position 307, Ala for the amino acid ofposition 380, and Ser for the amino acid of position 434, according toEU numbering, in the amino acid residues of the Fc region contained inthe antibody constant region of SEQ ID NO: 5, 6, 7, or 8. Anothernon-limiting embodiment of this Fc region includes Fc regions containingGln for the amino acid of position 307, Ala for the amino acid ofposition 380, and Ala for the amino acid of position 434. Furthermore,another non-limiting embodiment of this Fc region includes Fc regionscontaining Tyr for the amino acid of position 252, Pro for the aminoacid of position 308, and Tyr for the amino acid of position 434. Adifferent non-limiting embodiment of this Fc region includes Fc regionscontaining Asp for the amino acid of position 251, Gln for the aminoacid of position 307, and His for the amino acid of position 434.

Another non-limiting embodiment of the Fc region whose binding activityto FcRn in the acidic pH range is stronger than the binding activity ofthe starting Fc region of human IgG1 includes at least one or more aminoacid alterations selected from the group consisting of:

alteration of the amino acid of position 238 to Leu;alteration of the amino acid of position 244 to Leu;alteration of the amino acid of position 245 to Arg;alteration of the amino acid of position 249 to Pro;alteration of the amino acid of position 252 to Tyr;alteration of the amino acid of position 256 to Pro;alteration of the amino acid of position 257 to Ala, Ile, Met, Asn, Ser,or Val;alteration of the amino acid of position 258 to Asp;alteration of the amino acid of position 260 to Ser;alteration of the amino acid of position 262 to Leu;alteration of the amino acid of position 270 to Lys;alteration of the amino acid of position 272 to Leu or Arg;alteration of the amino acid of position 279 to Ala, Asp, Gly, His, Met,Asn, Gln, Arg, Ser, Thr, Trp, or Tyr;alteration of the amino acid of position 283 to Ala, Asp, Phe, Gly, His,Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr;alteration of the amino acid of position 285 to Asn;alteration of the amino acid of position 286 to Phe;alteration of the amino acid of position 288 to Asn or Pro;alteration of the amino acid of position 293 to Val;alteration of the amino acid of position 307 to Ala, Glu, or Met;alteration of the amino acid of position 311 to Ala, Ile, Lys, Leu, Met,Val, or Trp;alteration of the amino acid of position 312 to Pro;alteration of the amino acid of position 316 to Lys;alteration of the amino acid of position 317 to Pro;alteration of the amino acid of position 318 to Asn or Thr;alteration of the amino acid of position 332 to Phe, His, Lys, Leu, Met,Arg, Ser, or Trp;alteration of the amino acid of position 339 to Asn, Thr, or Trp;alteration of the amino acid of position 341 to Pro;alteration of the amino acid of position 343 to Glu, His, Lys, Gln, Arg,Thr, or Tyr;alteration of the amino acid of position 375 to Arg;alteration of the amino acid of position 376 to Gly, Ile, Met, Pro, Thr,or Val;alteration of the amino acid of position 377 to Lys;alteration of the amino acid of position 378 to Asp or Asn;alteration of the amino acid of position 380 to Asn, Ser, or Thr;alteration of the amino acid of position 382 to Phe, His, Ile, Lys, Leu,Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr;alteration of the amino acid of position 423 to Asn;alteration of the amino acid of position 427 to Asn;alteration of the amino acid of position 430 to Ala, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr;alteration of the amino acid of position 431 to His or Asn;alteration of the amino acid of position 434 to Phe, Gly, His, Trp, orTyr;alteration of the amino acid of position 436 to Ile, Leu, or Thr;alteration of the amino acid of position 438 to Lys, Leu, Thr, or Trp;alteration of the amino acid of position 440 to Lys; andalteration of the amino acid of position 442 to Lys;according to EU numbering, in the amino acid residues of the Fc regioncontained in the antibody constant region of SEQ ID NO: 5, 6, 7, or 8.The number of amino acids to be altered is not particularly limited andamino acids at only two sites may be altered and amino acids at three ormore sites may be altered.

Another non-limiting embodiment of the Fc region whose binding activityto FcRn in the acidic pH range is stronger than the binding activity ofthe starting Fc region of human IgG1 include Fc regions containing Ilefor the amino acid of position 257, and Ile for the amino acid ofposition 311, according to EU numbering, in the amino acid residues ofthe Fc region contained in the antibody constant region of SEQ ID NO: 5,6, 7, or 8. Another non-limiting embodiment of this Fc region includesFc regions containing Ile for the amino acid of position 257 and His forthe amino acid of position 434. Another non-limiting embodiment of thisFc region includes Fc regions containing Val for the amino acid ofposition 376 and His for the amino acid of position 434.

A non-limiting embodiment of the Fc region whose polynucleotide sequenceis linked to a polynucleotide encoding an antigen-binding domainincludes, for example, Fc regions having binding activity to human FcRnin the neutral pH range. Examples of Fc regions having binding activityto human FcRn in the neutral pH range include Fc regions in which atleast one or more amino acids at positions selected from the groupconsisting of positions 221-225, 227, 228, 230, 232, 233-241, 243-252,254-260, 262-272, 274, 276, 278-289, 291-312, 315-320, 324, 325,327-339, 341, 343, 345, 360, 362, 370, 375-378, 380, 382, 385-387, 389,396, 414, 416, 423, 424, 426-438, 440, and 442, according to EUnumbering, are substituted in the amino acid residues of the Fc regionincluded in the antibody constant region of SEQ ID NO: 5, 6, 7, or 8.

Another non-limiting embodiment of the aforementioned Fc region havingbinding activity to FcRn in the neutral pH range includes Fc regions inwhich amino acids at positions 237, 248, 250, 252, 254, 255, 256, 257,258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314,315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424,428, 433, 434, and 436, according to EU numbering, are substituted inthe amino acid residues of the Fc region contained in the antibodyconstant region of SEQ ID NO: 5, 6, 7, or 8. By substituting at leastone amino acid selected from these amino acids with a different aminoacid, the Fc region included in the antigen-binding molecule can bind tohuman FcRn in the neutral pH range.

Another non-limiting embodiment of the aforementioned Fc region havingbinding activity to FcRn in the neutral pH range includes Fc regionscontaining at least one or more amino acids selected from the groupconsisting of:

Met for the amino acid of position 237;Ile for the amino acid of position 248;Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr for the amino acid ofposition 250;Phe, Trp, or Tyr for the amino acid of position 252;Thr for the amino acid of position 254;Glu for the amino acid of position 255;Asp, Asn, Glu, or Gln for the amino acid of position 256;Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val for the amino acid ofposition 257;His for the amino acid of position 258:Ala for the amino acid of position 265;Ala or Glu for the amino acid of position 286;His for the amino acid of position 289;Ala for the amino acid of position 297;Ala for the amino acid of position 303;Ala for the amino acid of position 305;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Val, Trp, or Tyr for the amino acid of position 307;Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr for the amino acid of position308;Ala, Asp, Glu, Pro, or Arg for the amino acid of position 309;Ala, His, or Ile for the amino acid of position 311;Ala or His for the amino acid of position 312;Lys or Arg for the amino acid of position 314;Ala, Asp, or His for the amino acid of position 315;Ala for the amino acid of position 317;Val for the amino acid of position 332;Leu for the amino acid of position 334;His for the amino acid of position 360;Ala for the amino acid of position 376;Ala for the amino acid of position 380;Ala for the amino acid of position 382;Ala for the amino acid of position 384;Asp or His for the amino acid of position 385;Pro for the amino acid of position 386;Glu for the amino acid of position 387;Ala or Ser for the amino acid of position 389;Ala for the amino acid of position 424;Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val,Trp, or Tyr for the amino acid of position 428;Lys for the amino acid of position 433;Ala, Phe, His, Ser, Trp, or Tyr for the amino acid of position 434; andHis, Ile, Leu, Phe, Thr, or Val for the amino acid of position 436;according to EU numbering. The number of amino acids to be altered isnot particularly limited and an amino acid at only one site may bealtered or amino acids at two or more sites may be altered. Combinationsof these amino acid alterations include, for example, those described inTable 2-1 to 2-33.

A non-limiting embodiment of the Fc region whose polynucleotide sequenceis linked to a polynucleotide encoding an antigen-binding domainincludes, for example, Fc regions whose binding activity toward anactivating FcγR is lower than that of the native Fc region toward anactivating FcγR. Another non-limiting embodiment of the Fc regionpreferably includes, for example, Fc regions in which one or more aminoacids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325,328, and 329 according to EU numbering are altered to amino acids thatare different from those of the native Fc region of SEQ ID NO: 5, 6, 7,or 8. The alterations in the Fc region are not limited to the aboveexample, and they may be, for example, alterations such asdeglycosylation (N297A and N297Q), IgG1-L234A/L235A,IgG1-A325A/A330S/P331S, IgG1-C226S/C229S,IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-L234F/L235E/P331S,IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268QN309L/A330S/A331S,IgG4-L235A/G237A/E318A, and IgG4-L236E described in Cur. Opin. inBiotech. (2009) 20 (6), 685-691; alterations such as G236R/L328R,L235G/G236R, N325A/L328R, and N325L/L328R described in WO 2008/092117;amino acid insertions at positions 233, 234, 235, and 237 according toEU numbering; and alterations at the positions described in WO2000/042072.

Another non-limiting embodiment of the aforementioned Fc region whosebinding activity toward activating FcγR is lower than the bindingactivity of the native Fc region toward activating FcγR includes Fcregions comprising at least one or more amino acids selected from thegroup consisting of:

Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, orTrp for the amino acid at position 234;Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, orArg for the amino acid at position 235;Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, or Tyr for the amino acidat position 236;Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val,Tyr, or Arg for the amino acid at position 237;Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, or Arg for the aminoacid at position 238;Gln, His, Lys, Phe, Pro, Trp, Tyr, or Arg for the amino acid at position239;Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp,Tyr, or Val for the amino acid at position 265;Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, orTyr for the amino acid at position 266;Arg, His, Lys, Phe, Pro, Trp, or Tyr for the amino acid at position 267;Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val for the amino acid at position 269;Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val for the amino acid at position 270;Arg, His, Phe, Ser, Thr, Trp, or Tyr for the amino acid at position 271;Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid atposition 295;Arg, Gly, Lys, or Pro for the amino acid at position 296;Ala for the amino acid at position 297;Arg, Gly, Lys, Pro, Trp, or Tyr for the amino acid at position 298;Arg, Lys, or Pro for the amino acid at position 300;Lys or Pro for the amino acid at position 324;Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, or Val for theamino acid at position 325;Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Valfor the amino acid at position 327;Arg, Asn, Gly, His, Lys, or Pro for the amino acid at position 328;Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp,Tyr, Val, or Arg for the amino acid at position 329;Pro or Ser for the amino acid at position 330;Arg, Gly, or Lys for the amino acid at position 331; orArg, Lys, or Pro for the amino acid at position 332;according to EU numbering. The number of amino acids to be altered isnot particularly limited, and an amino acid at only one site may bealtered or amino acids at two or more sites may be altered.

In a non-limiting embodiment of the present invention, two polypeptidesforming an Fc region that are derived from a bispecific antibody asdescribed above can be suitably used as the Fc region to be included inan antigen-binding molecule. More specifically, it is preferable to usetwo polypeptides that constitute an Fc region, and which comprise Cysfor the amino acid at position 349 and Trp for the amino acid atposition 366 according to EU numbering in the amino acid sequence of oneof the polypeptides; and Cys for the amino acid at position 356, Ser forthe amino acid at position 366, Ala for the amino acid at position 368,and Val for the amino acid at position 407 as indicated by EU numberingin the amino acid sequence of the other polypeptide.

In another non-limiting embodiment of the present invention, twopolypeptides that constitute an Fc region and which comprises Asp forthe amino acid at position 409 according to EU numbering in the aminoacid sequence of one of the polypeptides, and Lys for the amino acid atposition 399 according to EU numbering in the amino acid sequence of theother polypeptide, may be suitably used as the Fc region. In the aboveembodiment, the amino acid at position 409 may be Glu instead of Asp,and the amino acid at position 399 may be Arg instead of Lys. Moreover,in addition to the amino acid Lys at position 399, Asp may suitably beadded as the amino acid at position 360 or Asp may suitably be added asthe amino acid at position 392.

In still another non-limiting embodiment of the present invention, twopolypeptides that constitute an Fc region and which comprise Glu for theamino acid at position 370 according to EU numbering in the amino acidsequence of one of the polypeptides, and Lys for the amino acid atposition 357 according to EU numbering in the amino acid sequence of theother polypeptide, may be suitably used as the Fc region.

In yet another non-limiting embodiment of the present invention, twopolypeptides that constitute an Fc region and which comprise Glu for theamino acid at position 439 according to EU numbering in the amino acidsequence of one of the polypeptides, and Lys for the amino acid atposition 356 according to EU numbering in the amino acid sequence of theother polypeptide, may be suitably used as the Fc region.

In still yet another non-limiting embodiment of the present invention,any of combinations of the above-mentioned embodiments, as shown below,may be suitably used as the Fc region:

(i) two polypeptides that constitute an Fc region and which comprise Aspfor the amino acid at position 409 and Glu for the amino acid atposition 370 according to EU numbering in the amino acid sequence of oneof the polypeptides, and Lys for the amino acid at position 399 and Lysfor the amino acid at position 357 according to EU numbering in theamino acid sequence of the other polypeptide (in this embodiment, theamino acid at position 370 according to EU numbering may be Asp insteadof Glu, and the amino acid Asp at position 392 according to EU numberingmay be used instead of the amino acid Glu at position 370 according toEU numbering);

(ii) two polypeptides that constitute an Fc region, and which compriseAsp for the amino acid at position 409 and Glu for the amino acid atposition 439 according to EU numbering of the amino acid sequence of oneof the polypeptides; and Lys for the amino acid at position 399 and Lysfor the amino acid at position 356 according to EU numbering in theamino acid sequence of the other polypeptide (in this embodiment, theamino acid Asp at position 360 according to EU numbering, the amino acidAsp at position 392 according to EU numbering, or the amino acid Asp atposition 439 according to EU numbering may be used instead of the aminoacid Glu at position 439 according to EU numbering);

(iii) two polypeptides that constitute an Fc region, and which compriseGlu for the amino acid at position 370 and Glu for the amino acid atposition 439 according to EU numbering in the amino acid sequence of oneof the polypeptides, and Ly for the amino acid at position 357 and Lysfor the amino acid at position 356 according to EU numbering in theamino acid sequence of the other polypeptide; or

two polypeptides that constitute an Fc region, and which comprise Aspthe amino acid at position 409, Glu for the amino acid at position 370,and Glu for the amino acid at position 439 according to EU numbering inthe amino acid sequence of one of the polypeptides; and Lys for theamino acid at position 399, Lys for the amino acid at position 357, andLys for the amino acid at position 356 according to EU numbering in theamino acid sequence of the other polypeptide (in this embodiment, theamino acid at position 370 according to EU numbering may not besubstituted with Glu, and furthermore, when the amino acid at position370 is not substituted with Glu, the amino acid at position 439 may beAsp instead of Glu, or the amino acid Asp at position 392 may be usedinstead of the amino acid Glu at position 439).

Further, in another non-limiting embodiment of the present invention,two polypeptides that constitute an Fc region and which comprise Lys forthe amino acid at position 356 according to EU numbering in the aminoacid sequence of one of the polypeptides, and Arg for the amino acid atposition 435 and Glu for the amino acid at position 439 according to EUnumbering in the amino acid sequence of the other polypeptide may alsobe suitably used.

In still another non-limiting embodiment of the present invention, twopolypeptides that constitute an Fc region and which comprise Lys for theamino acid at position 356 and Lys for the amino acid at position 357according to EU numbering in the amino acid sequence of one of thepolypeptides, and Glu for the amino acid at position 370, Arg for theamino acid at position 435, and Glu for the amino acid at position 439according to EU numbering in the amino acid sequence of the otherpolypeptide may also be suitably used.

Antigen-binding molecules of the present invention are isolated fromculture media of cells transformed with a desired expression vector inwhich a polynucleotide encoding an antigen-binding domain and apolynucleotide encoding a polypeptide containing an Fc region, whichhave been linked in the above-described manner, are operably linked.

When the Fc region contained in the antigen-binding molecule of thepresent invention is an Fc region that has been modified so that thepercentage of the Fc region to which a fucose-deficient sugar chain hasbeen attached, or bisecting N-acetylglucosamine has been attached, willbecome higher, the above-mentioned transformed host cells that aresuitably used are host cells that have low ability to add fucose to asugar chain as a result of modification of the activity to form thesugar chain structure of a polypeptide to be modified with a sugar chain(for example, WO 2000/061739, WO 2002/031140, and WO 2006/067913). In anon-limiting embodiment of such host cells, host cells deficient in theactivity of an enzyme or transporter selected from the group consistingof fucosyltransferase (EC 2.4.1.152), fucose transporter (SLC35C1), GMD(GDP-mannose-4,6-dehydratase) (EC 4.2.1.47), Fx(GDP-keto-6-deoxymannose-3,5-epimerase, 4-reductase) (EC 1.1.1.271), andGFPP (GDP-β-L-fucose pyrophosphorylase (EC 2.7.7.30), may be suitablyused (for example, WO 2000/061739, WO 2002/031140, and WO 2006/067913).Host cells deficient in such activity can be produced, for example, by amethod that destroys the genes of these functional proteins endogenousto CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63mouse myeloma cells, PER cells, PER.C6 cells, HEK293 cells, hybridomacells, or such so that they are unable to function.

When the Fc region contained in the antigen-binding molecule of thepresent invention is an Fc region having a sugar chain containing abisecting GlcNAc, the above-described transformed cells that aresuitably used are host cells expressing a gene encoding a functionalprotein having GnTIII (β-1,4-mannosyl-glycoprotein4-β-N-acetylglucosaminyltransferase) (EC2.4.1.144) activity or GalT(β-1,4-galactosyltransferase) (EC 2.4.1.38) activity to produceantibodies which have bisecting GlcNAc-containing sugar chains(WO2002/079255 and such). In another suitable non-limiting embodiment,host cells that co-express, in addition to the aforementioned functionalproteins, a gene encoding a functional protein having human ManII(manosidase II) (3.2.1.114) activity, a gene encoding a functionalprotein having GnTI (β-1,2-acetylglucosaminyltransferase I) (EC2.4.1.94) activity, a gene encoding a functional protein having GnTII(β-1,2-acetylglucosaminyltransferase II) (EC 2.4.1.143) activity, a geneencoding a functional protein having ManI (mannosidase) (EC 3.2.1.113)activity, and α-1,6-fucosyl transferase (EC 2.4.1.68), are suitably used(WO2004/065540).

Antigen-binding molecules of the present invention are produced usingmethods that follow the methods for producing antibodies, such asisolation from culture media of the above-mentioned cells, which aredescribed in the section “Antibodies” above. A non-limiting embodimentof the aforementioned polypeptides containing an Fc region includes, forexample, the antibody constant region of SEQ ID NO: 5, 6, 7, or 8. Anon-limiting embodiment of the antigen-binding molecules of the presentinvention is for example, a full-length antibody molecule.

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions comprising anantigen-binding molecule that does not act systemically in the blood ornormal tissues, but acts on lesions such as cancer and inflamed sites,to exhibit drug efficacy while avoiding side effects. Theantigen-binding molecule contained in the pharmaceutical composition ofthe present invention binds to an antigen expressed in cancer cells,immune cells, stromal cells, or such in cancer tissues; an antigensecreted in cancer tissues; or an antigen expressed by immune cells orsuch in inflammatory tissues; and an antigen secreted in inflammatorytissues; and cannot bind to antigens expressed in normal tissues;therefore, side effects due to cytotoxic activity, neutralizingactivity, or such against normal tissues are avoided; and at the sametime, potent cytotoxic effects, growth suppressing effects, andimmunity-enhancing action on cancers, or immunosuppressive effectsagainst inflammatory cells in inflammatory tissues, are exhibited. Forexample, a bispecific or biparatopic antigen-binding molecule containingan antigen-binding domain that binds to EGFR expressed on cancer cellsand an antigen-binding domain that binds to CD3 expressed on T cells ina manner dependent on a cancer tissue-specific compound, does not bindto EGFR expressed on normal tissues but bind to EGFR expressed on cancercells; thereby exhibiting potent antitumor effects while avoiding sideeffects. Specifically, while the antigen-binding molecule binds to CD3expressed on T cells in the vicinity of cancer cells in a mannerdependent on a cancer tissue-specific compound, the molecule does notbind to CD3 expressed on T cells that are not in the vicinity of cancercells. Therefore, the molecule activates T cells in the vicinity ofcancer cells, exhibiting potent antitumor effects while avoiding sideeffects.

Such antigen-binding molecules that bind to an antigen in target tissuesbut not in other normal tissues and blood exhibit drug efficacy whileavoiding side effects. Antigen-binding molecules provided by the presentinvention, which bind to an antigen by using a small molecule present athigh concentrations in target tissues in vivo as a switch, namely, smallmolecule switch antigen-binding molecules, do not bind to the antigen ina normal environment where the small molecule is not present, but canbind to the antigen in target tissues where the small molecule ispresent at high concentrations.

A non-limiting embodiment of such small molecule switch antigen-bindingmolecules includes cancer tissue-specific, or inflammatorytissue-specific, compound-dependent antigen-binding molecules; and acancer tissue-specific or inflammatory tissue-specific compound such asadenosine, adenosine 5′-triphosphate (ATP), inosine, kynurenine,prostaglandin E2 (PGE2), succinic acid, and lactic acid, which arepresent at a high concentration in cancer tissues or inflammatorytissues and capable of functioning as a switch, provides a switchfunction by being sandwiched between the antigen-binding molecule of thepresent invention (the paratope contained therein) and the antigen (theepitope contained therein). In the absence of the compound, theinteraction between the paratope in the antigen-binding molecule of thepresent invention and the epitope in the antigen is not sufficient forthe antigen-binding molecule of the present invention to be able to bindto the antigen. In the presence of the compound, the compound interposesbetween the paratope in the antigen-binding molecule of the presentinvention and the epitope in the antigen; and the antigen-bindingmolecule that has bound to the antigen in a target tissue such as cancertissue or inflammatory tissue, where the compound is present at a highconcentration, can exhibit drug efficacy on cells expressing theantigen. Moreover, since this binding of the switch compound isreversible, the binding of an antigen-binding molecule of the presentinvention to an antigen by means of these switch compounds may becontrolled in a reversible manner. Thus, antigen-binding molecules ofthe present invention which can exhibit drug efficacy in a lesion sitesuch as cancer tissue or inflammatory tissue by binding to pathogeniccells such as cancer cells or immune cells in a cancer tissue orinflammatory tissue or by binding to an antigen secreted in a cancertissue or inflammatory tissue are useful as pharmaceutical compositions.The pharmaceutical compositions of the present invention may comprise apharmaceutically acceptable carrier.

In the present invention, pharmaceutical compositions generally refer topharmaceutical agents for treating or preventing, or testing anddiagnosing diseases. Furthermore, in the present invention, the phrase“pharmaceutical composition containing an antigen-binding molecule whoseantigen-binding activity varies depending on the concentration of atarget tissue-specific compound” can be rephrased as “method fortreating a disease which comprises administering to a subject to betreated an antigen-binding molecule whose antigen-binding activityvaries depending on the concentration of a target tissue-specificcompound”, or rephrased as “use of an antigen-binding molecule whoseantigen-binding activity varies depending on the concentration of atarget tissue-specific compound in the production of a pharmaceuticalfor treating a disease”. Furthermore, the phrase “pharmaceuticalcomposition containing an antigen-binding molecule whose antigen-bindingactivity varies depending on the concentration of a targettissue-specific compound” can be rephrased as “use of an antigen-bindingmolecule whose antigen-binding activity varies depending on theconcentration of a target tissue-specific compound, for treating adisease”.

The pharmaceutical compositions of the present invention can beformulated by methods known to those skilled in the art. For example,they can be used parenterally, in the form of injections of sterilesolutions or suspensions including water or other pharmaceuticallyacceptable liquid. For example, such compositions can be formulated bymixing in the form of unit dose required in the generally approvedmedicine manufacturing practice, by appropriately combining withpharmacologically acceptable carriers or media, specifically withsterile water, physiological saline, vegetable oil, emulsifier,suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle,preservative, binder, or such. In such formulations, the amount ofactive ingredient is adjusted to obtain an appropriate amount in apre-determined range.

Sterile compositions for injection can be formulated using vehicles suchas distilled water for injection, according to standard formulationpractice. Aqueous solutions for injection include, for example,physiological saline and isotonic solutions containing dextrose or otheradjuvants (for example, D-sorbitol, D-mannose, D-mannitol, and sodiumchloride). It is also possible to use in combination appropriatesolubilizers, for example, alcohols (ethanol and such), polyalcohols(propylene glycol, polyethylene glycol, and such), non-ionic surfactants(polysorbate 80™, HCO-50, and such).

Oils include sesame oil and soybean oils. Benzyl benzoate and/or benzylalcohol can be used in combination as solubilizers. It is also possibleto combine buffers (for example, phosphate buffer and sodium acetatebuffer), soothing agents (for example, procaine hydrochloride),stabilizers (for example, benzyl alcohol and phenol), and/orantioxidants. Appropriate ampules are filled with the preparedinjections.

The pharmaceutical compositions of the present invention are preferablyadministered parenterally. For example, the compositions in the dosageform for injections, transnasal administration, transpulmonaryadministration, or transdermal administration are administered. Forexample, they can be administered systemically or locally by intravenousinjection, intramuscular injection, intraperitoneal injection,subcutaneous injection, or such.

Administration methods can be appropriately selected in consideration ofthe patient's age and symptoms. The dose of a pharmaceutical compositioncontaining an antigen-binding molecule can be, for example, from 0.0001to 1,000 mg/kg for each administration. Alternatively, the dose can be,for example, from 0.001 to 100,000 mg per patient. However, the presentinvention is not limited by the numeric values described above. Thedoses and administration methods vary depending on the patient's weight,age, symptoms, and such. Those skilled in the art can set appropriatedoses and administration methods in consideration of the factorsdescribed above.

Amino acids contained in the amino acid sequences of the presentinvention may be post-translationally modified (for example, themodification of an N-terminal glutamine into a pyroglutamic acid bypyroglutamylation is well-known to those skilled in the art). Naturally,such post-translationally modified amino acids are included in the aminoacid sequences in the present invention.

All prior art documents cited in this specification are incorporatedherein by reference.

Herein below, the present invention will be specifically described withthe Examples; however, the present invention should not be limitedthereto.

EXAMPLES Example 1 Concept of Antibodies that Bind to Antigens Via SmallMolecules Serving as a Switch, which are Present at High Concentrationsin Target Tissues

In order to exert drug efficacy while avoiding adverse effects, there isa need for drug discovery technology that works in lesions such ascancer or inflammatory sites without acting systemically in normaltissues or blood. Antibody molecules that can bind to antigens expressedon cancer cells but are incapable of binding to the antigens expressedon normal tissues after administration can exert strong cytotoxiceffects against cancer while avoiding adverse effects on normal tissuesas a result of cytotoxic action. For example, antigen-binding moleculesthat have been altered from the above-described EGFR-BiTE (Non-patentDocument 9), which cannot bind to EGFR expressed on normal tissues butare capable of binding to EGFR expressed on cancer cells, can exertstrong an antitumor effect while avoiding adverse effects. Meanwhile,BiTE exerts an antitumor effect by recruiting and activating T cells viaCD3 (Non-patent Document 8); and if it is possible to confer EGFR-BiTEwith the property of binding to CD3 expressed on T cells in the vicinityof cancer cells but not to CD3 expressed on T cells outside the vicinityof cancer cells, EGFR-BiTE altered to have the property can activate Tcells in cancer and thus can exert strong antitumor effects whileavoiding adverse effects.

However, this is not limited to only antibody pharmaceuticals againstcancer. When an antibody molecule binds and inhibits cytokines in thesynovial fluid of inflamed joints in rheumatoid arthritis but does notsystemically inhibit the cytokines, the molecule can exert potenttherapeutic effects against inflammatory/autoimmune diseases such asrheumatoid arthritis while avoiding increased risks of infection due tosystemic neutralization of cytokines.

As described above, antibodies that bind to antigens in cancer tissuesbut not to antigens in other tissues such as normal tissues and bloodcan exert drug efficacy while avoiding adverse effects. However, idealantibodies having such properties have not been reported so far.Meanwhile, as shown in FIG. 1, antibody molecules that bind to antigensvia small molecules, as a switch, that are present at highconcentrations in cancer tissues in vivo (i.e., small molecule switchantibodies), do not bind to antigens in environments in the absence ofsuch small molecules; and they can bind to antigens in target tissueswhere the small molecules are present at high concentrations.

In developing such small-molecule switch antibodies, first it was tosearch for small molecules that are present at high concentration incancer tissues and are considered to be usable as a switch. The resultsuggested that adenosine, adenosine triphosphate (adenosine5′-triphosphate (ATP)), inosine, kynurenine, prostaglandin E2 (PGE2),succinic acid, and lactic acid were promising as a switch. Each of thesesmall molecules is either produced by cancer cells, or released fromcancer cells after cell death, or produced by immune cells etcinfiltrating cancer tissues, and thus they are present at highconcentrations in cancer tissues; however, they are present at lowerconcentrations in normal tissues and blood in comparison to cancertissues. If these small molecules can be sandwiched in a complex betweenan antibody and an antigen in such a way as shown in FIG. 2, the smallmolecules can achieve the switch function. Specifically, in the absenceof small molecules, the antigen-antibody interaction is insufficient andthe antibody cannot bind to its antigen. Meanwhile, in the presence of asmall molecule, the antibody can bind to its antigen via the smallmolecule sandwiched between the antibody and antigen. In other words, inthe presence of a low concentration of small molecules, theantigen-antibody interaction is insufficient and the antibody cannotbind to its antigen, while in the presence of a high concentration ofsmall molecules, the antibody can bind to its antigen via a smallmolecule sandwiched between the antibody and antigen. Furthermore, thebinding of small molecules as a switch is reversible, and the regulationof antigen binding by the small molecule switch is also reversible.

In this context, first, the present inventors attempted to isolate smallmolecule switch antibodies against IL-6 (Br. J. Haematol. (2011) 152(5), 579-92) which is reported to be involved in cancer cell growth.

Example 2 Acquisition of Antibodies that Bind to Human IL-6 in thePresence of Small Molecules from a Human Antibody Library UsingPhage-Display Techniques

(2-1) Construction of a Phage-Display Library of Naïve Human Antibodies

A phage-display library of human antibodies consisting of multiplephages that present the Fab domains of human antibodies whose sequenceswere different from one another was constructed using as a template,polyA RNA prepared from human PBMC, commercially available human polyARNA, or such according to a method known to those skilled in the art.

(2-2) Acquirement of Antibodies that Bind to Human IL-6 in the Presenceof Small Molecules from the Library by Bead Panning

The phage-display library of naïve human antibodies constructed asdescribed in (2-1) was screened for antibodies that exhibitantigen-binding activity in the presence of small molecules,specifically, by collecting phages displaying antibodies that in thepresence of small molecules exhibit antigen-binding activity to antigenscaptured by beads. Phages were collected from a phage suspension elutedfrom the beads in the absence of small molecules. In this preparationmethod, the antigen used was biotin-labeled human IL-6.

Phages produced in E. coli containing the phagemid vector constructedfor phage display were purified by a conventional method. Then, a phagelibrary suspension was prepared by dialyzing the phages against TBS.Next, BSA was added at a final concentration of 4% to the phage librarysuspension. Panning was performed using antigen-immobilized magneticbeads. The magnetic beads used were NeutrAvidin coated beads (Sera-MagSpeedBeads NeutrAvidin-coated) or Streptavidin coated beads (DynabeadsM-280 Streptavidin).

To efficiently isolate small molecule switch antibodies which depend onsmall molecules which can serve as a switch in cancer tissues, panningwas carried out to enrich antibodies that bind to antigens in thepresence of a mixed solution of small molecules (adenosine, adenosinetriphosphate (adenosine 5′-triphosphate (ATP)), inosine, kynurenine,prostaglandin E2 (PGE2), succinic acid, and lactic acid (hereinafterreferred to as small molecule cocktail (SC)) but not in the absence ofSC.

Specifically, together with 250 pmol of biotin-labeled antigen, SCcontaining adenosine triphosphate sodium salt (ATP-Na), adenosine,inosine, succinic acid, and lactic acid at a final concentration of 1mM, prostaglandin E2 (PGE2) at a final concentration of 1 μM, andkynurenine at a final concentration of 100 μM, which had been adjustedto be pH 7.4 with NaOH, was contacted with the prepared phage librarysuspension for 60 minutes at room temperature. Then, BSA-blockedmagnetic beads were added to the phage library suspension, and theantigen-phage complex was allowed to bind to the magnetic beads at roomtemperature for 15 minutes. After washing once with SC/TBS (TBScontaining SC), the beads were combined with 0.5 ml of 1 mg/ml trypsinsolution. Immediately after suspending the beads at room temperature for15 minutes, the phage suspension was collected from the isolated beadsusing a magnetic stand. The collected phage suspension was added to 10ml of E. coli cells of strain ER2738 at the logarithmic growth phase(OD600=0.4 to 0.7). The E. coli was infected with the phage byincubating the above E. coli with gently stirring at 37° C. for onehour. The infected E. coli was seeded in a 225 mm×225 mm plate. Then,phages were collected from the culture medium of the seeded E. coli toprepare a liquid stock of phage library.

The first round of panning was carried out to collect phages that arecapable of binding in the presence of small molecules, while the secondand subsequent rounds of panning were performed to enrich phages thatare capable of binding to antigens in the presence of SC. Specifically,the prepared phage library suspension was mixed with 40 pmolbiotin-labeled antigen, SC, and NaOH, and contacted with the smallmolecules and antigens for 60 minutes at room temperature. BSA-blockedmagnetic beads were added and allowed to bind to the antigen-phagecomplex for 15 minutes at room temperature. The beads were washed with 1ml of SC/TBST and SC/TBS. Then, immediately after 0.5 ml of TBS wasadded to suspend the beads at room temperature, a phage suspension wascollected from the isolated beads using a magnetic stand. After thistreatment was repeated, the two separately eluted phage suspensions weremixed together. Then, the resultant beads were combined with 0.5 ml ofTBS and stirred at room temperature for five minutes. A phage suspensionwas collected from the isolated beads using a magnetic stand. Byaddition of 5 μl of 100 mg/ml trypsin to the collected phage suspension,the pIII protein (helper phage-derived protein pIII) that does notdisplay Fab was cleaved off from phages, and the ability of phages thatdo not display Fab to infect E. coli was eliminated. The phagescollected from the trypsinized phage suspension were added to 10 ml ofE. coli strain ER2738 at the logarithmic growth phase (OD600=0.4 to0.7). The E. coli was incubated at 37° C. for one hour under gentlestirring to infect phage. The infected E. coli was seeded in a 225mm×225 mm plate. The two types of infected E. coli obtained through thesecond round of panning were mixed in equal amounts at this time point.Then, phages were collected from the culture medium of the seeded E.coli to prepare a phage library suspension. Panning was performed threetimes to isolate antibodies that have antigen-binding activity in thepresence of SC.

(2-3) Acquisition of Antibodies that Bind to Human IL-6 in the Presenceof Small Molecules from the Library Using a Negative Selection Method

The constructed phage-display library of naïve human antibodies wasscreened for antibodies that exhibit antigen-binding activity in thepresence of small molecules. As a first step of screening, thephage-display library of naïve human antibodies was contacted withbiotin-labeled antigen-streptavidin in the absence of small molecules toeliminate phages displaying antibodies that have antigen-bindingactivity even in the absence of small molecules. Then, panning wasperformed in the presence of small molecules in the same manner. Thus,screening was carried out for antibodies that have antigen-bindingactivity in the presence of small molecules. Biotin-labeled IL-6 wasused as the antigen.

Phages were produced in E. coli retaining the constructed phage-displayphagemid. The produced phages were purified by a conventional method,and then a phage library suspension was prepared by dialyzing the phagesagainst TBS. Then, BSA was added to the phage library suspension at afinal concentration of 4%. The magnetic beads used were NeutrAvidincoated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) or Streptavidincoated beads (Dynabeads M-280 Streptavidin). Panning was performed usingantigen-immobilized magnetic beads.

Together with 250 pmol of biotin-labeled antigen, SC containing ATP-Na,adenosine, inosine, succinic acid, and lactic acid at a finalconcentration of 1 mM, PGE2 at a final concentration of 1 μM, andkynurenine at a final concentration of 100 μM, whose pH was adjusted to7.4 with NaOH, was added and contacted with the prepared phage librarysuspension for 60 minutes at room temperature. Then, BSA-blockedmagnetic beads were added to the phage library suspension, and theantigen-phage complex was allowed to bind to the magnetic beads at roomtemperature for 15 minutes. After washing once with SC/TBS, the beadswere combined with 0.5 ml of 1 mg/ml trypsin solution. Immediately aftersuspending the beads at room temperature for 15 minutes, the phagesuspension was collected from the isolated beads using a magnetic stand.The collected phage suspension was added to 10 ml of E. coli cells ofstrain ER2738 at the logarithmic growth phase (OD600=0.4 to 0.7). The E.coli was incubated at 37° C. for one hour with gentle stirring to beinfected by phage. The infected E. coli was seeded in a 225 mm×225 mmplate. Then, phages were collected from the culture medium of the seededE. coli to prepare a liquid stock of phage library.

The first round of panning was carried out to collect phages that arecapable of binding in the presence of SC, while the second andsubsequent rounds of panning were performed to enrich phages that arecapable of binding to antigens in the presence of SC. Specifically, 250pmol of biotinylated antigen was added to BSA-blocked Sera-MagNeutrAvidin beads for binding at room temperature for 15 minutes. Thebeads were washed three times with TBS. The phage library suspensionsubjected to BSA blocking was added to the beads, and allowed to bindthereto at room temperature for one hour. Phages that did not bind tothe antigens or beads were collected by isolating the beads using amagnetic stand. 40 pmol of biotin-labeled antigen, SC, and NaOH wereadded to the collected phages. Thus, the phage library was contactedwith the small molecules in SC at room temperature for 60 minutes. Then,BSA-blocked magnetic beads were added to the mixture of labeled antigen,SC, and phage library, and allowed to bind to the antigen-phage complexfor 15 minutes at room temperature. The beads were washed with 1 ml ofSC/TBST and SC/TBS. Then, 0.5 ml of 1 mg/ml trypsin solution was addedto the mixture. After the mixed suspension was stirred at roomtemperature for 20 minutes, phages were collected from the beads thathad been separated using a magnetic stand. The collected phages wereadded to 10 ml of E. coli strain ER2738 at the logarithmic growth phase(OD600=0.4 to 0.7). The E. coli was incubated at 37° C. for one hourunder gentle stirring to be infected by phage. The infected E. coli wasseeded in a 225 mm×225 mm plate. Panning was performed three times toisolate antibodies that have antigen-binding activity in the presence ofSC.

(2-4) Assessment of Binding Activity in the Presence of Small Moleculesby Phage ELISA

Culture supernatants containing phages were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained by the method described above. Thecollected culture supernatants were treated by ultrafiltration usingNucleoFast 96 (MACHEREY-NAGEL). 100 μl of the collected culturesupernatants were added to each well of NucleoFas96 and centrifuged(4500 g for 45 minutes) to remove the flow-through portion. 100 μl ofH₂O was added to each well, and again the NucleoFast 96 was centrifuged(4500 g for 30 minutes) for washing. After 100 μl of TBS was added, theNucleoFast 96 was allowed to stand for five minutes at room temperature.Finally, a phage suspension was collected from the supernatant in eachwell.

After addition of TBS or SC/TBS, the purified phages were subjected toELISA by the following procedure. A StreptaWell 96 microtiter plate(Roche) was coated overnight with 100 μl of TBS containing thebiotin-labeled antigen. After the antigen was removed by washing eachwell of the plate with TBST, the wells were blocked with 250 μl of 2%skim milk-TBS for one hour or more. 2% skim milk-TBS was removed, andthen the prepared, purified phages were added to each well. The platewas allowed to stand at 37° C. for one hour to allow binding ofantibody-displaying phages to the antigen in the presence or absence ofSC in each well. After each well was washed with TBST or SC/TBST, theHRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) dilutedwith TBS or SC/TBS was added thereto, and the plate was incubated forone hour. Following washing with TBST or SC/TBST, the TMB singlesolution (ZYMED) was added to each well, and the chromogenic reaction inthe solution was terminated by adding sulfuric acid. Then, the developedcolor was assessed by measuring absorbance at 450 nm.

Phage ELISA of isolated 96 clones revealed a clone “I6NMSC1-3_A11”,which has binding activity to human IL-6 as an antigen in the presenceof a small molecule cocktail.

Example 3 Assessment of Antibodies that Bind to Antigens in the Presenceof Small Molecules

(3-1) Expression and Purification of Antibodies that Bind to Human IL-6

Genes were amplified from clone I6NMSC1-3_A11 which had been assessed tohave antigen-binding activity in the presence of SC using specificprimers (SEQ ID NOs: 110 and 112) by phage ELISA as described in Example2. The nucleotide sequences of the genes were analyzed (the heavy chainand light chain sequences are shown in SEQ ID NOs: 30 and 31,respectively). The gene encoding the variable region of I6NMSC1-3_A11was inserted into an animal expression plasmid for human IgG1/Lambda,while each of the genes encoding the variable regions of knownanti-human IL-6 antibody CLB8-F1 (the heavy chain and light chain areSEQ ID NOs: 32 and 33, respectively) and the variable regions ofanti-human glypican 3 antibody GC413 (the heavy chain and light chainare SEQ ID NOs: 34 and 35, respectively) as a negative control wereinserted into an animal expression plasmid for human IgG1/kappa.Antibodies were expressed using the method described below. FreeStyle293-F (Invitrogen) which is derived from human fetal kidney cells weresuspended at a cell density of 1.33×10⁶ cells/ml in FreeStyle 293Expression Medium (Invitrogen) and aliquoted at 3 ml into each well of a6-well plate. The plasmid DNA was transfected into the cells bylipofection. From the culture supernatants after four days of culture ina CO₂ incubator (37° C., 8% CO₂, 90 rpm), antibodies were purified by amethod known to those skilled in the art using rProtein A Sepharose™Fast Flow (Amersham Biosciences). Absorbance of solutions of purifiedantibodies was measured at 280 nm using a spectrophotometer. From thevalues obtained by measurement, the concentrations of purifiedantibodies were calculated using an extinction coefficient determined bythe PACE method (Protein Science (1995) 4, 2411-2423).

(3-2) Identification of Small Molecules Necessary for Human IL-6 Bindingof the Obtained Antibodies

Three types of antibodies: isolated I6NMSC1-3_A11 (hereinafterabbreviated as A11), and CLB8-F1 and GC413 as controls were subjected toELISA under the nine conditions described in Table 3. Meanwhile, eachsmall molecule was appropriately prepared at the concentrations shown inTable 3 using the buffers indicated in Table 4. Biotin-labeled humanIL-6 was used as the antigen.

TABLE 3 Condition Small molecule Concentration 1 ATP-Na 1 mM 2 Adenosine1 mM 3 Inosine 1 mM 4 PGE2  1 μM 5 Succinic acid 1 mM 6 Lactic acid 1 mM7 Kynurenine 100 μM 8 ATP 1 mM, Adenosine 1 mM, Inosine 1 mM, PGE2 1 μM,Succinic acid 1 mM, Lactic acid 1 mM, Kynurenine 100 μM 9 — —

TABLE 4 Wash buffer 10 mM ACES, 150 mM NaCl, 0.05% Tween20, pH7.4Blocking Buffer 10 mM ACES, 150 mM NaCl, 2% BSA, pH7.4 Sample Buffer 10mM ACES, 150 mM NaCl, Each small molecule, pH7.4

First, a StreptaWell 96 microtiter plate (Roche) was coated at roomtemperature for one hour or more with 100 μl of PBS containing thebiotin-labeled antigen. After washing with the Wash buffer to removeunbound antigen from the plate, each well was blocked for one hour ormore with 250 μl of the Blocking Buffer. The Blocking Buffer was removedfrom each well. The purified IgGs were prepared to 2.5 μg/ml in a SampleBuffer containing small molecules at the final concentrations shown inTable 3, and each was aliquoted at 100 μl to each well of the plate. Theplate was allowed to stand at room temperature for one hour to allowbinding of each IgG to the antigen in each well. After washing with aWash Buffer containing the small molecules at the final concentrationsshown in Table 3, an HRP-conjugated anti-human IgG antibody (BIOSOURCE)diluted with a Sample Buffer containing the same small molecules wasadded to each well. The plate was incubated for one hour. Following washwith a Wash Buffer containing each small molecule, the TMB singlesolution (ZYMED) was added to each well. The chromogenic reaction in thesolution of each well was terminated by adding sulfuric acid. Then, thedeveloped color was assessed by measuring absorbance at 450 nm.

The measurement result is shown in FIG. 3. The result showed that theabsorbance of CLB8-F1 was constant regardless of the type or presence ofsmall molecule, whereas the absorbance of I6NMSC1-3_A11 was markedlylower under condition 9 (without small molecules) as compared to undercondition 8 (the complete small molecule cocktail solution). Similar tophage ELISA, this result showed that I6NMSC1-3_A11 had the property thatits antigen binding is altered depending on the presence of smallmolecules. Meanwhile, I6NMSC1-3_A11 showed equivalent absorbance undercondition 7 (in the presence of 100 μM kynurenine) to that undercondition 8; however, the absorbance was markedly lower under otherconditions. This result demonstrates that I6NMSC1-3_A11 is an antibodythat binds to human IL-6 as an antigen in the presence of kynurenine butnot in the absence of kynurenine.

Example 4 Assessment of the Effect of Kynurenine on Human IL6 Binding bySurface Plasmon Resonance (4-1) Assessment of Kynurenine for its SwitchFunction in Human IL-6 Binding

Using Biacore T200 (GE Healthcare), A11 was analyzed for its interactionwith human IL-6 (Kamakura Techno-Science, Inc.) in antigen-antibodyreaction. Sensor chip CM5 (GE Healthcare) was immobilized with anappropriate amount of protein A/G (Invitrogen) by amine coupling.Antibodies of interest were captured by the chip to allow interaction toIL-6 as an antigen. The two types of running buffers used were 10 mmol/lACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, 100 μmol/l kynurenine, pH7.4, and 10 mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4.The interaction with IL-6 as an antigen was assessed at 37° C. Thebuffer used to dilute IL-6 was the same running buffer as describedabove.

A diluted solution of human IL-6 and a running buffer as a blank wereinjected at a flow rate of 5 μl/min for three minutes to allowinteraction of human IL-6 with A11 captured on the sensor chip. Then,the running buffer was injected at a flow rate of 5 μl/min for threeminutes. After observation of human IL-6 dissociation from the antibody,10 mmol/l glycine-HCl (pH 1.5) was injected at a flow rate of 30 μl/minfor 30 seconds to regenerate the sensor chip. The dissociation constantK_(D) (M) of A11 was calculated for human IL-6 based on the associationrate constant ka (1/Ms) and dissociation rate constant kd (1/s), both ofwhich are kinetic parameters calculated from the sensorgram obtained bythe measurement. Each parameter was calculated using the Biacore T200Evaluation Software (GE Healthcare).

The sensorgrams for the interaction between A11 and 4 μmol/l human IL-6obtained by the measurement in the presence or absence of 100 μmol/lkynurenine are shown in FIG. 4. As shown in FIG. 4, A11 bound to IL-6 inthe presence of 100 μmol/l kynurenine; however, in the absence ofkynurenine, the IL-6 binding was undetectable. This demonstrates thatA11 has the property that it binds to IL-6 via kynurenine as a switch.Meanwhile, the dissociation constant K_(D) of A11 was 1.0E⁻⁶ mol/l inthe presence of 100 μmol/l kynurenine.

(4-2) Assessment for the Effect of Kynurenine Concentration on HumanIL-6 Binding

Then, the effect of kynurenine concentration on antigen-antibodyreaction between A11 and human IL-6 was assessed using Biacore T200 (GEHealthcare). The running buffer used was 10 mmol/l ACES, 150 mmol/lNaCl, 0.05% (w/v) Tween20, pH 7.4. Antigen-antibody reaction between A11and human IL-6 was assessed at 25° C. A11 was immobilized onto sensorchip CM5 by amine coupling, and as an analyte IL-6 was diluted to 1μmol/l with 10 mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4containing kynurenine at various concentrations, and allowed to interactfor 60 seconds to observe changes in the amount of binding. The resultis shown in FIG. 5. This result demonstrated that the higher theconcentration of kynurenine as a switch, the more IL-6 binds to A11.

Next, the same experiment as described above was carried out to assessthe effect of kynurenine concentration on antigen-antibody reactionbetween human IL-6 and the human IL-6-binding antibody H01 (the heavychain and light chain are SEQ ID NOs: 36 and 37, respectively)immobilized on sensor chip CM5, which was derived from a library andserved as a control for the switch function of kynurenine in A11. Theresult is shown in FIG. 6. This result confirmed that for the controlanti-IL-6 antibody H01 derived from a library, its binding to IL-6 isnot altered even if the kynurenine concentration changes.

Then, the effect of the concentration of kynurenine as a switch in thedivalent binding of A11 to IL-6 was assessed using Biacore T200 (GEHealthcare). The running buffer used was 10 mmol/l ACES, 150 mmol/lNaCl, 0.05% (w/v) Tween20, pH 7.4. Antigen-antibody reaction between A11and human IL-6 was assessed at 25° C. IL-6 was immobilized onto sensorchip CM5 by amine coupling, and as an analyte A11 was diluted to 0.1μmol/l with 10 mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4containing various concentrations of kynurenine, and allowed to interactfor 60 seconds to observe changes in the amount of divalent binding ofA11 to IL-6. The result is shown in FIG. 7. In this assay system, A11 isexpected to bind in a divalent manner since IL-6 is immobilized on asensor chip. With such an assay system where A11 recognizes IL-6 in adivalent manner, the amount of A11 bound to IL-6 was also observed toincrease with a higher kynurenine concentration. This resultdemonstrated that A11 has the property that in its divalent binding, italso binds to IL-6 via kynurenine as a switch.

(4-3) Effect of Kynurenine as a Switch on the Dissociation of Antibodiesfrom Human IL-6

Using Biacore T200 (GE Healthcare), A11 bound to IL-6 in the presence ofkynurenine was tested to assess whether in the absence of kynurenine, itdissociates in a kynurenine concentration-dependent manner. The runningbuffer used was 10 mmol/l ACES, 150 mmol/1 NaCl, 0.05% (w/v) Tween20, pH7.4, and 10 mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4,100 μmol/l kynurenine. Assay was carried out at 25° C. IL-6 wasimmobilized onto sensor chip CM5 by amine coupling, and as an analyteA11 was diluted to 0.1 μmol/l with 10 mmol/l ACES, 150 mmol/l NaCl,0.05% (w/v) Tween20, pH 7.4, containing 100 μmol/l kynurenine, andallowed to interact for 60 seconds. Then, the dissociation of IL-6 wasmonitored with each type of running buffer. In order to compare thedegree of dissociation between respective running buffer conditions, theamounts of IL-6 bound to A11 were normalized and compared by taking thevalue in the presence of 100 μmol/l kynurenine as 100. A sensorgramrepresenting the interaction between A11 and IL-6 after normalization isshown in FIG. 8. The result shown in FIG. 8 demonstrates that A11 hasthe property that it binds to IL-6 in the presence of kynurenine andthen rapidly dissociates from IL-6 in the absence of kynurenine.Specifically, the kynurenine-mediated regulation of the antibody bindingto human IL-6 by kynurenine was demonstrated to be completelyreversible.

These results demonstrated that A11 is an antibody that binds to IL-6 inthe presence of kynurenine via kynurenine as a switch, but isdissociated from IL-6 in the absence of kynurenine. It was alsoconfirmed that it is possible to have full ON/OFF regulation of A11 sothat it has no human IL-6-binding activity in the absence of kynurenine.The switch function was expected to be achieved in the manner such asshown in FIG. 2.

(4-4) Assessment of Kynurenine for its Binding to Human IL-6

The interaction between IL-6 (Kamakura Techno-Science, Inc.) andkynurenine was analyzed using Biacore T200 (GE Healthcare). Sensor chipCM5 (GE Healthcare) was immobilized with about 5000 RU of IL-6 by aminecoupling, and 800, 400, 200, 100, 50, or 25 nmol/l kynurenine wasallowed to interact with IL-6. The running buffer used was 10 mmol/1ACES, 150 mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4. A11 measurements forthe interaction described above were carried out at 25° C. Kynureninewas diluted using the running buffer. The obtained sensorgram showingthe interaction between IL-6 and kynurenine is shown in FIG. 9.

About 5000 RU of IL-6 was immobilized in the above-described experiment.The molecular weights of IL-6 and kynurenine were about 20000 g/mol andabout 200 g/mol, respectively. Thus, at maximum about 50 RU ofkynurenine was expected to interact. Under the measurement conditiondescribed above, however, obvious interaction with IL-6 was notdetectable even when kynurenine was allowed to interact at a maximalconcentration of 800 nmol/l.

Based on the result of the Example described above, the KD of kynureninefor formation of the complex consisting of A11, IL-6, and kynurenine isestimated to be several tens nM to several nM. This also suggests thatif hypothetically kynurenine interacts directly with IL-6, theinteraction would be observed unambiguously when kynurenine was allowedto interact at 800 nmol/l. The result described above implies thepossibility that kynurenine does not interact directly with IL-6 butinteracts with A11 or the A11-IL-6 complex at several tens nM.

Example 5 Acquisition of Anti-Adenosine Antibodies by Rabbit B CellCloning (5-1) Design of Immunogen to Construct Adenosine-Binding Library

The immunogens used in immunizing rabbits were 2′-Adenosine-PEG-Tetanustoxin p30 helper peptide (2′-Adenosine-PEG-peptide) shown in FIG. 10 and5′-Adenosine-PEG-Tetanus toxin p30 helper peptide(5′-Adenosine-PEG-peptide) shown in FIG. 11. The Tetanus toxin p30helper peptide consists of the amino acid sequence FNNFTVSFWLRVPKVSASHLE(SEQ ID NO: 4), and is a peptide identified as an epitope of T cellreceptor expressed on helper T cells (Eur. J. Immunol. (1989) 19,2237-2242). The peptide is known to activate antibody production (J.Immunol. (1992) 149, 717-721). When linked to adenosine, the peptideserves as an adjuvant and thus is expected to enhance the production ofantibodies against adenosine. The linkage between adenosine and theTetanus toxin p30 helper peptide was designed to be through PEG so thatepitopes of antibodies against adenosine can hardly contain the Tetanustoxin p30 helper peptide. Adenosine is an ATP metabolite, and since thephosphate groups of ATP are attached to the 5′ hydroxyl group ofadenosine, antibodies that do not recognize the 5′ hydroxyl group ofadenosine as an epitope may also bind to ATP in addition to adenosine.That is, it would be easier to obtain antibodies that can bind to bothadenosine and ATP by using as an immunogen the 5′-Adenosine-PEG-Tetanustoxin p30 helper peptide, while it would be easier to obtain antibodiesthat bind to adenosine but not to ATP by using as an immunogen the2′-Adenosine-PEG-Tetanus toxin p30 helper peptide. For this reason, thetwo types of immunogens which contain the Tetanus toxin p30 helperpeptide linked to the 2′ or 5′ position of adenosine were prepared inthe manner described in (5-2). In addition, 2′-Adenosine-PEG-biotin(FIG. 12) and 5′-Adenosine-PEG-biotin (FIG. 13), in which biotin isconjugated instead of the Tetanus toxin p30 helper peptide, wereproduced as described below. By assessing the binding to these two typesof Adenosine-PEG-biotin, antibodies can be tested to demonstrate thattheir epitopes do not contain the Tetanus toxin p30 helper peptide.

(5-2) Synthesis of Immunogens to Prepare Adenosine-Binding Library

2′-Adenosine-PEG-peptide (adenosine 2′-PEG-peptide conjugate or2′-(PEG-peptide)adenosine) and 2′-Adenosine-PEG-biotin (adenosine2′-PEG-biotin conjugate or 2′-(PEG-biotin)adenosine) were synthesized inthe manner described below. The synthesized 2′-Adenosine-PEG-peptide and2′-Adenosine-PEG-biotin were analyzed or fractionated under theconditions below.

The conditions of LCMS analysis are noted as below.

TABLE 5 Column Analysis Column Flow rate temperature condition Apparatus(length, mm) Mobile phase Gradient (A/B) (ml/min) (° C.) WavelengthSQDAA05 Acquity UPLC/SQD Aldrich Ascentis Express A) 10 mM AcONH4, 95/5=> 0/100 1.0 35 210-400 nm C18 (2.1 × 50) H2O (1.0 min) => 0/100 PDAtotal B) MeOH (0.4 min) SQDAA50 Acquity UPLC/SQD Aldrich AscentisExpress A) 10 mM AcONH4, 50/50 => 0/100 1.0 35 210-400 nm C18 (2.1 × 50)H2O (0.7 min) => 0/100 PDA total B) MeOH (0.7 min) SQDFA05 AcquityUPLC/SQD Aldrich Ascentis Express A) 0.1% FA, H2O 95/5 => 0/100 1.0 35210-400 nm C18 (2.1 × 50) B) 0.1% FA CH3CN (1.0 min => 0/100 PDA total(0.4 min) SQDFA50 Acquity UPLC/SQD Aldrich Ascentis Express A) 0.1% FA,H2O 50/50 => 0/100 1.0 35 210-400 nm C18 (2.1 × 50) B) 0.1% FA CH3CN(0.7 min) => 0/100 PDA total (0.7 min)

The conditions of preparative HPLC are described as below.

TABLE 6 Column Preparative Column Flow rate temperature conditionApparatus (length, mm) Mobile phase Gradient (A/B) (ml/min) (° C.)Wavelength A Preparative HPLC Aldrich Ascentis RP- A) 0.1% FA H2Oisocratic (A/B): 15/85 20.0 40 254, 258 nm system with Amide B) 0.1% FAMeCN injection/fractionation (21.2 × 150 mm 5 μm) (Gilson, Inc.) BPreparative HPLC YMC Actus ODS-A A) 20 mM AcONH4, isocratic (A/B): 47/5320.0 40 254, 258 nm system with (20 × 100 mm 5 μm) H2Oinjection/fractionation B) 20 mM AcONH4 (Gilson, Inc.) MeOH/MeCN (1/1)(5-2-1) Synthesis of Compound 006 (Boc-Phe-Asn-Asn-Phe-Thr (tBu)-Val-Ser(tBu)-Phe-Trp (Boc)-Lue-Arg (Pbf)-Val-Pro-Lys (Boc)-Val-Ser(tBu)-Ala-Ser (tBu)-His (Trt)-Leu-Glu (tBu)-OH)

Peptide synthesis was performed by the Fmoc method using a peptidesynthesizer (Multipep RS; Intavis). All Fmoc amino acids were purchasedfrom WATANABE CHEMICAL INDUSTRIES, LTD. The detailed procedure of thetreatment was in the manual attached to the synthesizer.

Fmoc-Glu(tBu)-OH linked at its C terminus to 2-chlorotrityl resin (250mg/column, 30 columns, 11.7 mmol), an N,N-dimethylformamide solutioncontaining various Fmoc amino acids (0.6 mol/l) and1-hydroxy-7-azabenzotriazole (0.375 mol/l), and an N,N-dimethylformamidesolution (10% v/v) of diisopropylcarbodiimide were loaded in thesynthesizer. The synthesis reaction was performed using as anFmoc-deprotection solution, an N,N-dimethylformamide solution (20% v/v)containing piperidine and 5% (wt/v) urea. After the resin was washedwith N,N-dimethylformamide, Fmoc deprotection was carried out, followedby one cycle of Fmoc amino acid condensation reaction. This cycle wasrepeated to elongate peptides on the resin surface. After elongation,the resin was washed with trifluoroethanol. Peptides were cleaved offfrom the resin by adding trifluoroethanol/dichloromethane (=1/1). Thus,compound 006 (7.2 g) was obtained as a crude product.

LCMS(ESI)m/z=1185(M+3H)3+

Retention time: 1.24 minute (Analysis condition, SQDAA05)

(5-2-2) Synthesis of Compound 007

A suspension of adenosine (2.00 g, 7.48 mmol) in N,N-dimethylformamide(40 ml) was cooled down to 0° C., and 60% sodium hydride (0.42 g, 10.48mol) was added thereto. The reaction mixture was stirred for one hour at0° C. After adding methyl bromoacetate (0.76 ml, 8.01 mmol), theresulting reaction mixture was stirred for five hours at roomtemperature, and acetic acid (1 ml) and methanol (3 ml) were addedthereto. The reaction mixture was concentrated under reduced pressure.The resulting residue was purified by normal phase silica gel columnchromatography (dichloromethane/methanol). Thus, compound 007 (0.93 g,37%) was obtained.

LCMS(ESI) m/z=340(M+H)+

Retention time: 0.27 minute (Analysis condition, SQDFA05)

(5-2-3) Synthesis of Compound 008

t-Butyldimethylsilyl chloride (999 mg, 6.63 mol) and imidazole (722 mg,10.61 mol) were added to a pyridine solution (8 ml) of compound 007 (900mg, 2.65 mmol). The reaction mixture was stirred for four hours at roomtemperature, and extracted with ethyl acetate/water. The extractedorganic layer was washed with a saturated sodium chloride solution, anddried over anhydrous sodium sulfate. After filtration, the organic layerwas concentrated under reduced pressure. The resulting residue waspurified by normal phase silica gel column chromatography(dichloromethane/methanol). Thus, compound 008 (1.17 g, 78%) wasobtained.

LCMS(ESI)m/z=568(M+H)+

Retention time: 1.10 minute (Analysis condition, SQDFA05)

(5-2-4) Synthesis of Compound 009

Lithium hydroxide (61 mg, 2.55 mol) dissolved in water (0.17 ml) wasadded to a solution of compound 008 (290 mg, 0.511 mmol) in methanol(0.34 ml)/tetrahydrofuran (0.34 ml). The reaction mixture was stirredfor 30 minutes at room temperature. The mixture was neutralized with 1 Mhydrochloric acid, and concentrated under reduced pressure. Theconcentrated residue was extracted with ethyl acetate/water. Theresulting organic layer was washed with a saturated sodium chloridesolution, and dried over anhydrous sodium sulfate. After filtration, theorganic layer was concentrated under a reduced pressure. Thus, compound009 (319 mg, 90%) was obtained.

LCMS(ESI)m/z=552(M−H)−

Retention time: 0.97 minute (Analysis condition, SQDFA05)

(5-2-5) Synthesis of Compounds 010 and 011

1-Hydroxybenzotriazole (75 mg, 0.553 mol) and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (106 mg,0.553 mol) were added to an N,N-dimethylformamide (1.5 ml) solution ofcompound 009 (255 mg, 0.460 mmol), and it was stirred for three minutesat room temperature. O-(2-aminoethyl)-O′-2-azidoethyl) nonaethyleneglycol (291 mg, 0.553 mmol) was added to the reaction mixture, and itwas stirred for three hours at room temperature. The reaction mixturewas concentrated under a reduced pressure, and the resulting residue waspurified by reverse phase silica gel column chromatography (aqueous 10mM ammonium acetate solution/methanol. Compounds 010 (177 mg, 4%) and011 (72 mg, 19%) were obtained.

Compound 010

LCMS(ESI)m/z=1063(M+H)+

Retention time: 0.98 minute (Analysis condition, SQDFA05)

Compound 011

LCMS(ESI)m/z=949(M+H)+

Retention time: 0.67 minute (Analysis condition, SQDFA05)

(5-2-6) Synthesis of Compound 012

10% palladium carbon (34 mg) was added to a solution of compound 010(170 mg, 0.160 mmol) in ethanol (1 ml). The reaction mixture was stirredfor two hours under hydrogen atmosphere, and again 10% palladium carbon(34 mg) was added thereto. The reaction mixture was stirred for twohours under a hydrogen atmosphere to complete the reaction. The filtrateof the reaction solution was concentrated under a reduced pressure.Compound 012 (34 mg, 95%) was obtained.

LCMS(ESI)m/z=1037(M+H)+

Retention time: 0.70 minute (Analysis condition, SQDFA05)

(5-2-7) Synthesis of Compounds 013 and 014

Compound 006 (354 mg, 0.110 mmol), 1-hydroxybenzotriazole (13 mg, 0.100mol), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(19 mg, 0.100 mol) were added to a solution of compound 012 (86 mg,0.083 mmol) in N,N-dimethylformamide (1.5 ml), and it was stirred fortwo hours at room temperature. The filtrate of the reaction mixture waspurified by preparative condition A described in Table 6. A mixture ofcompounds 013 and 014 (72 mg) was obtained.

Compound 013

LCMS(ESI)m/z=1525(M+3H)3+, 1144(M+4H)4+

Retention time: 1.13 minute (Analysis condition, SQDAA50)

Compound 014

LCMS(ESI)m/z=1444(M+3H)3+, 1083(M+4H)4+

Retention time: 1.02 minute (Analysis condition, SQDAA50)

(5-2-8) Synthesis of 2′-Adenosine-PEG-Peptide (Adenosine 2′-PEG-PeptideConjugate or 2′-(PEG-Peptide)Adenosine) (Compound 015)

Trifluoroacetic acid (16 ml), dichloromethane (8 ml), water (1.3 ml),and tetraisopropylsilane (1.3 ml) were added to the mixture of compounds013 and 014 (42 mg), and it was stirred for six hours at roomtemperature. The residue obtained by concentrating the reaction mixtureunder reduced pressure was purified by preparative condition B describedin Table 6. Thus, compound 015 (10 mg) was obtained.

LCMS(ESI)m/z=1090(M+3H)3+, 818(M+4H)4+

Retention time: 0.52 minute (Analysis condition, SQDAA50)

(5-2-9) Synthesis of Compound 016

10% palladium carbon (34 mg) was added to a solution of compound 011 (70mg, 0.074 mmol) in ethanol (1 ml), and the reaction mixture was stirredfor five hours under hydrogen atmosphere. The filtrate of the reactionmixture was concentrated under reduced pressure. Thus, compound 016 (58mg, 85%) was obtained.

LCMS(ESI)m/z=923(M+H)+

Retention time: 0.50 minute (Analysis condition, SQDFA05)

(5-2-10) Synthesis of Compound 017

D-biotin N-succinimidyl (24 mg, 0.069 mmol) and triethylamine (13 μl,0.094 mol) were added to a solution of compound 016 (58 mg, 0.063 mmol)in N,N-dimethylformamide (1 ml), and it was stirred for two hours atroom temperature. Then, after D-biotin N-succinimidyl (5 mg, 0.015 mmol)was added, the reaction was completed upon 1.5 hours of stirring at roomtemperature. The reaction mixture was purified by reverse phase silicagel column chromatography (aqueous 10 mM ammonium acetatesolution/methanol. Compound 017 (50 mg, 69%) was obtained.

LCMS(ESI)m/z=1149(M+H)+

Retention time: 1.04 minute (Analysis condition, SQDFA05)

(5-2-11) Synthesis of 2′-Adenosine-PEG-Biotin (Adenosine 2′-PEG-BiotinConjugate or 2′-(PEG-Biotin)Adenosine) (Compound 018)

A solution of 1 M tetra-n-butylammonium fluoride in tetrahydrofuran (65μl, 0.065 mmol) was added to a solution of compound 017 (62 mg, 0.054mmol) in tetrahydrofuran (2 ml), and it was stirred at room temperaturefor one hour. Then, 1 M tetra-n-butylammonium fluoride intetrahydrofuran solution (20 μl, 0.020 mmol) was added, and the reactionwas completed by stirring at room temperature for one hour. The reactionmixture was concentrated under a reduced pressure, and the residue waspurified by reverse phase silica gel column chromatography (aqueous 0.1%formic acid solution/0.1% formic acid in acetonitrile). Compound 018 (12mg, 21%) was obtained.

LCMS(ESI)m/z=1035(M+H)+

Retention time: 0.71 minute (Analysis condition, SQDAA05)

Furthermore, 5′-Adenosine-PEG-peptide and 5′-Adenosine-PEG-biotin werealso synthesized by the same reaction.

(5-3) Production of Adenosine-Binding Antibodies in Animals and AntibodyScreening

Rabbits were immunized with 2′-Adenosine-PEG-peptide and/or5′-Adenosine-PEG-peptide by a conventional method. Candidates for cellswith adenosine-binding activity were selected from suspensions of cellscollected from blood of the immunized rabbits, by using autoMACS ProSeparator and FACSAria (BD) which uses Adenosine-PEG-biotin-bindingactivity and rabbit IgG expression as indicators. Then, screening wascarried out with antibodies secreted in the culture supernatants of theselected cells. In the screening, ELISA was performed to assess thepresence of binding activity to Adenosine-PEG-biotin. ELISA was alsoperformed to assess whether adenosine, when added in combination withAdenosine-PEG-biotin at a level 1000 times or more of that ofAdenosine-PEG-biotin, suppresses the binding to Adenosine-PEG-biotin.The H-chain and L-chain variable regions were isolated by PCR from cellsselected using as an indicator the presence of theAdenosine-PEG-biotin-binding activity as well as suppression of thebinding to Adenosine-PEG-biotin by adenosine added in combination withAdenosine-PEG-biotin. The obtained variable regions were expressed incombination with a human IgG1 heavy chain constant region and a humanlight chain constant region.

(5-4) Acquisition of B Cells to Prepare Adenosine-Binding Immune Library

Cells were collected from the spleens of rabbits immunized with the2′-Adenosine-PEG-Tetanus toxin peptide and 5′-Adenosine-PEG-Tetanustoxin peptide. From the cell suspensions, candidates for cells withadenosine-binding activity were selected using autoMACS Pro Separatorand FACSAria (BD) with the presence of binding to Adenosine-PEG-biotinas well as the expression of rabbit IgG or IgM as indicators. Theselected cells were washed with PBS(−), and the prepared cell pelletswere used to construct immune libraries.

Example 6 Assessment of Clones Obtained by Rabbit B Cell Cloning

(6-1) Assessment of Clones Obtained by Rabbit B Cell Cloning for theirBinding Activity to 2′-Adenosine-PEG-Biotin

Clones obtained by rabbit B cell cloning were assessed for their bindingactivity to adenosine by the SPR method. Antigen-antibody reactionbetween the clones and 2′-Adenosine-PEG-Biotin was kinetically analyzedusing Biacore 4000 (GE Healthcare). Sensor chip CM5 (GE Healthcare) wasimmobilized with an appropriate amount of protein A/G (Invitrogen) byamine coupling. Antibodies of interest were captured by the chip. Then,after 100 nmol/12′-adenosine-PEG-Biotin was interacted as an analyte for60 seconds, the dissociation of the analyte was monitored and measuredfor 60 seconds. The running buffer used was HBS-P+(GE Healthcare). Allmeasurements were carried out at 25° C. The analyte was diluted usingthe running buffer.

The respective antibodies were compared for their binding activity to2′-Adenosine-PEG-Biotin using as an indicator the value(N_binding_(—)100) of dividing the amount of binding upon interactionwith 2′-Adenosine-PEG-Biotin by the amount of capture (RU) for eachantibody, and the value (N_stability_(—)100) of dividing the amount ofdissociation of 2′-Adenosine-PEG-Biotin from each antibody for 60seconds after interaction with 2′-Adenosine-PEG-Biotin by the amount ofcapture (RU) for each antibody. Regarding antibodies for which theamount of capture was 1500 RU or less, their binding was notsufficiently detectable and thus they were excluded from the subjects tobe tested. The result is shown in FIG. 14. The result shown in FIG. 14demonstrates that the B cell cloning method yielded adenosine-bindingclones with various affinity.

(6-2) Assessment of 2′-Adenosine-PEG-Biotin-Binding Clones for theirBinding Activity to Adenosine and ATP, and Sequence Analysis of theClones

Clones which were demonstrated to bind to 2′-Adenosine-PEG-Biotin wereassessed for their binding to adenosine and ATP by SPR and competitiveELISA.

(6-2-1) Assessment by SPR of 2′-Adenosine-PEG-Biotin-Binding Clones fortheir Binding to Adenosine and ATP

Using Biacore T200 (GE Healthcare), antibodies SMB0002, SMB0089, andSMB0104 obtained by the B cell cloning method were analyzed for theirinteraction with adenosine and ATP in antigen-antibody reaction. Sensorchip CM5 (GE Healthcare) was immobilized with an appropriate amount ofprotein A/G (Invitrogen) by amine coupling. Antibodies of interest werecaptured by the chip to allow interaction to adenosine or ATP as anantigen. The running buffer used was 10 mmol/l ACES, 150 mmol/lNaCl,0.05% (w/v) Tween20, pH 7.4. All measurements were carried out at 25° C.The antigens were diluted using the running buffer.

Regarding SMB0002, SMB0089, and SMB0104, the diluted antigen solutionsand the running buffer as a blank were injected at a flow rate of 20μl/min for two minutes to allow interaction of each antigen with theantibodies captured on the sensor chip. Then, the running buffer wasinjected at a flow rate of 20 μl/min for three minutes, and dissociationof the antigens from the antibodies was observed. Next, 10 mmol/lglycine-HCl (pH 1.5) was injected at a flow rate of 30 μl/min for 30seconds to regenerate the sensor chip. The association rate constant ka(1/Ms) and dissociation rate constant kd (1/s), both of which arekinetic parameters, were calculated from the sensorgrams obtained by themeasurement. The dissociation constant KD (M) was calculated based onthese constants. Each parameter was calculated using the Biacore T200Evaluation Software (GE Healthcare).

The result showed that multiple clones including SMB0002, SMB0089, andSMB0104 bound to both adenosine and ATP. The sensorgrams observed toassess each clone for its binding at adenosine concentrations of 500,125, 31.3, and 7.81 nM or at ATP concentrations of 5000, 1250, 313, and78.1 nM are shown in FIG. 15. As shown in FIG. 15, SMB0002, SMB0089, andSMB0104 were demonstrated to bind to both adenosine and ATP. KDs ofSMB0002, SMB0089, and SMB0104 were 9.3E⁻⁹, 6.9E⁻⁹, 4.1E⁻⁸ (mol/l), and1.0E⁻⁵ (mol/l) for adenosine, and 1.0E⁻⁵, 8.8E⁻⁷, and 1.4E⁻⁷ (mol/l) forATP, respectively.

In the same manner using Biacore 4000 (GE Healthcare), antibody SMB0171obtained by B cell cloning was analyzed for its interaction withadenosine and ATP in antigen-antibody reaction. Sensor chip CM5 (GEHealthcare) was immobilized with an appropriate amount of protein A/G(Invitrogen) by amine coupling. Antibodies of interest were captured bythe chip to allow interaction with adenosine or ATP as an antigen. Therunning buffer used was HBS-P+(GE Healthcare). All measurements werecarried out at 25° C. The antigens were diluted using the runningbuffer.

Regarding SMB0171, diluted antigen solutions and the running buffer as ablank were injected at a flow rate of 10 μl/min for one minute to allowinteraction of each antigen with antibodies captured on the sensor chip.Then, the running buffer was injected at a flow rate of 10 μl/min forthree minutes, and dissociation of the antibody from the antigens wasobserved. Then, 10 mmol/l glycine-HCl (pH 1.5) was injected at a flowrate of 30 μl/min for 30 seconds to regenerate the sensor chip. Theassociation rate constant ka (1/Ms) and dissociation rate constant kd(1/s), both of which are kinetic parameters, were calculated from thesensorgrams obtained by measurement. The dissociation constant KD (M)was calculated based on these constants. Each parameter was calculatedusing the Biacore 4000 Evaluation Software (GE Healthcare).

The result showed that SMB0171 binds to ATP. The sensorgrams observed inassessing each clone for its binding at ATP concentrations of 50 and 5μM are shown in FIG. 16. As shown in FIG. 16, SMB0171 was demonstratedto bind to ATP. KD of SMB0171 for ATP was 5.9E⁻⁶ (mol/l).

(6-2-2) Assessment of 2′-Adenosine-PEG-Biotin-Binding Clones for theirBinding to Adenosine and ATP by Competitive ELISA

Antibodies demonstrated to bind to 2′-Adenosine-PEG-Biotin were dilutedto 1 μg/ml with PBS, and added to each well of a 384-well MAXISorp(Nunc). To immobilize the antibodies, the plate was allowed to stand forone hour or more at room temperature. After the antibodies diluted withPBS were removed from each well, TBS containing 1% BSA was added theretoand the plate was allowed to stand for one hour or more. Then, the TBS(pH 7.4) containing 1% BSA was removed from the plate.2′-Adenosine-PEG-Biotin diluted to 50 nM with PBS, a mixture of2′-Adenosine-PEG-Biotin and adenosine diluted to 50 nM and 500 μMrespectively with PBS, a mixture of 2′-Adenosine-PEG-Biotin and ATPdiluted to 50 nM and 500 μM respectively with PBS, or PBS alone wasadded to the plate. The plate was allowed to stand at room temperaturefor one hour, and then washed three times with 80 μl of PBS containing0.05% Tween-20. Then, Streptavidin-HRP (Thermo fisher scientific)diluted 20000 times with PBS was added to each well, and the plate wasallowed to stand for one hour or more at room temperature. After theplate was washed three times with 80 μl of PBS containing 0.05%Tween-20, a chromogenic substrate (ABTS peroxidase substrate) was addedto each well. After the plate was incubated for one hour, colordevelopment in the solution of each well was assessed by measuringabsorbance at 405 nm using SpectraMax from Molecular Device.

As shown in FIG. 17, the result showed that the binding of SMB0002 to2′-Adenosine-PEG-Biotin was inhibited by adding excess amounts ofadenosine and ATP. Thus, the antibody clones were demonstrated to bindnot only to 2′-Adenosine-PEG-Biotin but also to both adenosine and ATP.

(6-2-3) Sequence Analysis of Adenosine- and ATP-Binding Clones by SPR

The amino acid sequences of clones demonstrated to bind to bothadenosine and ATP are shown in Table 7.

TABLE 7 Clone name Heavy chain SEQ ID NO Light chain SEQ ID NO SMB0002SEQ ID NO: 38 SEQ ID NO: 39 SMB0089 SEQ ID NO: 40 SEQ ID NO: 41 SMB0104SEQ ID NO: 42 SEQ ID NO: 43 SMB0171 SEQ ID NO: 44 SEQ ID NO: 45

Example 7 Acquisition of Antibodies that Bind to Adenosine and/or ATPfrom Human Antibody Library Using Phage Display Techniques

(7-1) Construction of Phage-Display Library of Naïve Human Antibodies

A phage-display library of human antibodies consisting of multiplephages that present the Fab domains of human antibodies whose sequenceswere different from one another was constructed using, as a template,polyA RNA prepared from human PBMC, commercially available human polyARNA, or such according to a method known to those skilled in the art.

(7-2) Acquisition of Antibodies that Bind to Adenosine and/or ATP fromLibrary by Bead Panning

The phage-display library of naïve human antibodies constructed asdescribed in (7-1) was screened for antibodies that exhibitantigen-binding activity, specifically, by collecting phages thatdisplay antibodies with binding activity to antigens captured by beads.Biotinylated ATP, 2′-adenosine-PEG-Biotin, and 5′-adenosine-PEG-Biotinwere used as antigens.

Phages produced in E. coli containing the phagemid vector constructedfor phage display were purified by a conventional method, and thendialyzed against TBS to prepare a phage library suspension. Then, BSAwas added at a final concentration of 4% to the suspension. Panning wasperformed using antigen-immobilized magnetic beads. The magnetic beadsused were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) and Streptavidin coated beads (Dynabeads M-280Streptavidin).

Next, 250 pmol of biotinylated ATP, 2′-adenosine-PEG-Biotin, and5′-adenosine-PEG-Biotin were added to the prepared phage librarysuspension. Thus, the phage library suspension was contacted withadenosine and ATP for 60 minutes at room temperature. Then, BSA-blockedmagnetic beads were added to the phage library suspension, and thecomplex of phage with adenosine and/or ATP was allowed to bind to themagnetic beads at room temperature for 15 minutes. The beads were washedonce with TBS. Then, the beads were combined with 0.5 ml of 1 mg/mltrypsin solution. Immediately after the beads were suspended at roomtemperature for 15 minutes, a phage suspension was collected from theisolated beads using a magnetic stand. The collected phage suspensionwas added to 10 ml of E. coli cells of strain ER2738 at the logarithmicgrowth phase (OD600=0.4 to 0.7). The E. coli was incubated at 37° C. forone hour under gentle stirring to be infected by phage. The infected E.coli was seeded in a 225 mm×225 mm plate. Then, phages were collectedfrom the culture medium of the seeded E. coli to prepare a liquid stockof phage library.

A second round of panning was performed to also enrich phages that arecapable of binding to adenosine and/or ATP. The prepared phage librarysuspension was contacted with adenosine and ATP for 60 minutes at roomtemperature by adding 50 pmol each of biotinylated ATP,2′-Adenosine-PEG-Biotin, and 5′-Adenosine-PEG-Biotin. Then, theBSA-blocked magnetic beads were added to the phage library suspension,and the complex of phage with adenosine and/or ATP was allowed to bindto the magnetic beads at room temperature for 15 minutes. The beads werewashed three times with TBST and twice with TBS. Then, the beads werecombined with 0.5 ml of 1 mg/ml trypsin solution. Immediately after thebeads were suspended at room temperature for 15 minutes, a phagesuspension was collected from the isolated beads using a magnetic stand.The collected phage suspension was added to 10 ml of E. coli cells ofstrain ER2738 at the logarithmic growth phase (OD600=0.4 to 0.7). The E.coli was incubated at 37° C. for one hour with gentle stirring to beinfected by phage. The infected E. coli was seeded in a 225 mm×225 mmplate. Then, phages were collected from the culture medium of the seededE. coli to prepare a liquid stock of phage library.

By the same procedure, panning was performed three times to obtainantibodies that are capable of binding to adenosine and/or ATP. In thefourth round of panning, TBST wash and TBS wash were each performed fivetimes.

(7-3) Assessment of Adenosine- and ATP-Binding Activity by Phage ELISA

From single colonies of E. coli obtained by panning as described in theExample above, culture supernatants containing phages were collectedaccording to a conventional method (Method Mol. Biol. (2002) 178,133-145). The collected culture supernatants were treated byultrafiltration using NucleoFast 96 (MACHERY-NAGEL). 100 μl of theculture supernatants were added to each well of NucleoFast 96 andcentrifuged at 4500 g for 45 minutes to remove flow through. Afteraddition of 100 μl of H₂O, the NucleoFast 96 was washed bycentrifugation at 4500 g for 30 minutes. After addition of 100 μl ofTBS, the NucleoFast 96 was allowed to stand for five minutes at roomtemperature. Then, phage suspensions were collected from thesupernatants.

Purified phages, to which TBS was added, were subjected to ELISA by thefollowing procedure. A StreptaWell 96 microtiter plate (Roche) wascoated at room temperature for one hour with 100 μl of TBS containingbiotin-labeled antigens (a mixture of equal amounts of2′-adenosine-PEG-biotin, 5′-adenosine-PEG-biotin, and ATP-PEG-biotin).After antigens were removed from each well of the plate by washing withTBST (TBS containing 0.1% Tween20), the wells were blocked with 250 μlof 2% skim milk/TBS for one hour or more. 2% skim milk/TBS was removed,and then the prepared, purified phages were added to each well. Theplate was allowed to stand at room temperature for one hour to allow thephage-displayed antibody to bind antigens in each well. After washingwith TBST, an HRP-conjugated anti-M13 antibody (Amersham PharmaciaBiotech) diluted with TBS was added to each well. The plate wasincubated for one hour. Following TBST wash, the TMB single solution(ZYMED) was added to each well. The chromogenic reaction in the solutionof each well was terminated by adding sulfuric acid. Then, the developedcolor was assessed by measuring absorbance at 450 nm.

From the 192 clones subjected to phage ELISA, 106 clones that have theability to bind to any one or two, or all three of2′-Adenosine-PEG-biotin, 5′-Adenosine-PEG-biotin, and ATP-PEG-biotinwere obtained.

Next, for the purpose of confirming to which antigen of2′-adenosine-PEG-biotin, 5′-adenosine-PEG-biotin, and ATP-PEG-biotinthese clones have binding ability, the purified phages diluted with TBSwere subjected to ELISA by the following procedure. A StreptaWell 96microtiter plate (Roche) was coated at room temperature for one hourwith 100 μl of TBS containing a biotin-labeled antigen(2′-adenosine-PEG-biotin, 5′-adenosine-PEG-biotin, or ATP-PEG-biotin).After the antigens were removed by washing each well of the plate withTBST, the wells were blocked with 250 μl of 2% skim milk/TBS for onehour or more. 2% skim milk/TBS was removed, and then the prepared,purified phages were added to each well. The plate was allowed to standat room temperature for one hour to allow binding of antibody-displayingphages to antigens in each well. After TBST wash, an HRP-conjugatedanti-M13 antibody (Amersham Pharmacia Biotech) diluted with TBS wasadded to each well. The plate was incubated for one hour. Following TBSTwash, the TMB single solution (ZYMED) was added to each well. Thechromogenic reaction in the solution of each well was terminated byadding sulfuric acid. Then, the developed color was assessed bymeasuring absorbance at 450 nm. The result of phage ELISA is shown inTable 8 below.

TABLE 8 Antigen binding ability Enrichment indicator (S/N ratio > 1.5)Number of panning  4 Number of clones subjected to ELISA 192 Number ofCombination of 2′-Adenosine-PEG- 106 ELISA- biotin,5′-Adenosine-PEG-biotin, and positive ATP-PEG-biotin clones2′-Adenosine-PEG-biotin  0 5′-Adenosine-PEG-biotin  6 ATP-PEG-biotin  76Bind to two or more of 2′-Adenosine-  1 PEG-biotin,5′-Adenosine-PEG-biotin, and ATP-PEG-biotin

Among the clones subjected to phage ELISA, a clone was demonstrated tobind to two or more types of antigens. Its gene was amplified withspecific primers using the antibody fragment as a template. Thenucleotide sequence of the gene was analyzed. This clone had the abilityto bind to both 5′-Adenosine-PEG-biotin and ATP-PEG-biotin, and wasnamed ATNLSA1-4_D12. The heavy-chain variable region sequence ofantibody ATNLSA1-4_D12 is shown in SEQ ID NO: 46, and its light-chainvariable region sequence is shown in SEQ ID NO: 47.

(7-4) Assessment of Adenosine- and ATP-Binding Activity by CompetitivePhage ELISA

Based on the structures of 5′-Adenosine-PEG-biotin and ATP-PEG-biotin,there remained the possibility that clone ATNLSA1-4_D12 (heavy chainvariable region, SEQ ID NO: 46; light chain, SEQ ID NO: 47), which wasdemonstrated by the result of phage ELISA to have the ability to bind toboth 5′-Adenosine-PEG-biotin and ATP-biotin, recognizes the biotin tagor PEG moiety. Thus, to demonstrate that ATNLSA1-4_D12 is not anantibody that recognizes the biotin tag or PEG, whether the antigenbinding is inhibited by adenosine or ATP was tested by phage ELISA usingATNLSA1-4_D12, and IL-6R-binding clone PF1 (heavy chain, SEQ ID NO: 48;light chain, SEQ ID NO: 49) prepared as a negative control.ATNLSA1-4_D12 and PF1 were each diluted with TBS and subjected to ELISAby the following procedure.

A StreptaWell 96 microtiter plate (Roche) was coated at room temperaturefor one hour with 100 μl of TBS containing biotin-labeled antigens (amixture of 5′-adenosine-PEG-biotin and ATP-PEG-biotin). After theantigens were removed by washing each well of the plate with TBST, thewells were blocked with 250 μl of 2% skim milk/TBS for one hour or more.2% skim milk/TBS was removed, and then the prepared, purified phageswere added to each well. The plate was allowed to stand at roomtemperature for one hour to allow binding of the antibody-displayingphages to the antigens in each well. Then, TBS that does not containantigen or that contains serial dilutions of ATP from an equal amount upto 10000 times that of the antigen was added to the wells. For thecompetition of the immobilized antigen with ATP, the plate was allowedto stand at room temperature for one hour. Then, after TBST wash, anHRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) dilutedwith TBS was added to each well. The plate was incubated for one hour.Following TBST wash, the TMB single solution (ZYMED) was added to eachwell. The chromogenic reaction in the solution of each well wasterminated by adding sulfuric acid. Then, the developed color wasassessed by measuring absorbance at 450 nm.

The measurement result is shown in FIG. 18. It was demonstrated that thehigher the ATP concentration, the smaller the degree of colordevelopment for ATNLSA1-4_D12 in the presence of an excess amount ofATP. Thus, the binding between ATNLSA1-4_D12 and its antigen wasdemonstrated to be inhibited in an ATP concentration-dependent manner.Meanwhile, in a control experiment with PF1 as a negative control, itsantigen binding was not detected regardless of the ATP concentration.The above finding demonstrates that ATNLSA1-4_D12 is an antibody thathas the ability to bind to ATP but does not recognize the biotin tag orPEG.

(7-5) Expression and Purification of Antibodies that Bind to ATP andAdenosine

Using specific primers, genes were amplified from clone ATNLSA1-4_D12that had been assessed to have binding activity to ATP and adenosine bythe phage ELISA described in Example 7. The nucleotide sequences of thegenes were analyzed (the heavy-chain and light-chain sequences are shownin SEQ ID NOs: 46 and 47, respectively). The gene encoding the variableregion of ATNLSA1-4_D12 was inserted into an animal expression plasmidfor human IgG1/Lambda. The antibody was expressed using the methoddescribed below. Cells of human fetal kidney cell-derived FreeStyle293-F (Invitrogen) were suspended at a cell density of 1.33×10⁶ cells/mlin FreeStyle 293 Expression Medium (Invitrogen) and aliquoted at 3 mlinto each well of a 6-well plate. The constructed plasmid was introducedinto the cells by lipofection. After four days of culture in a CO₂incubator (37° C., 8% CO₂, 90 rpm), the antibody was purified from theculture supernatants by a method known to those skilled in the art usingrProtein A Sepharose™ Fast Flow (Amersham Biosciences). Absorbance ofthe purified antibody solutions was measured at 280 nm using aspectrophotometer. From the values obtained by the measurement, theconcentration of the purified antibody was calculated using anextinction coefficient determined by the PACE method (Protein Science(1995) 4, 2411-2423).

(7-6) Assessment of the ATP- and Adenosine-Binding Antibody for its ATPand Adenosine Binding by Surface Plasmon Resonance

Biacore T200 (GE Healthcare) was used to analyze the interaction of D12,in which the constant region of IgG is linked to the variable region ofclone ATNLSA1-4_D12 with ATP- and adenosine-binding activity, inantigen-antibody reaction. Sensor chip CM5 or CM4 (GE Healthcare) wasimmobilized with an appropriate amount of protein A (Life technologies)by amine coupling. The antibody of interest was captured by the chip toallow interaction with ATP (Wako), adenosine (Wako), or ADP (adenosinediphosphate) (Wako) as an antigen. The running buffer used was 50 mMTris-HCl (Takara, T903), 500 mM NaCl, 0.01% (w/v) Tween20. The antigenwas allowed to interact for 30 seconds at a flow rate of 30 μl/min, andwas dissociated for 30 seconds. The interaction with the antigen wasassessed at 15° C. The antigen was diluted using the same runningbuffer.

The dissociation constant K_(D) (M) was calculated based on theassociation rate constant ka (1/Ms) and dissociation rate constant kd(1/s), both of which are kinetic parameters calculated from thesensorgram obtained by the measurement. Alternatively, the dissociationconstant K_(D) (M) was calculated using steady state analysis. Eachparameter was calculated using the Biacore T200 Evaluation Software (GEHealthcare).

To calculate the K_(D) for adenosine, the binding response was assessedat various concentrations of adenosine in the presence or absence of 20μmol/l ADP. In addition, the binding response was separately assessed inthe presence of 20 μmol/l ADP. The response (R) for specific adenosinebinding was obtained by subtracting the value of binding response in thepresence of ADP alone from the binding response to variousconcentrations of adenosine in the presence of ADP, and then subtractingthe resultant value, which is assumed to correspond to the non-specificbinding components, from the value of binding response to adenosine inthe absence of ADP. From a curve in which adenosine concentration isplotted on the X axis and R calculated according to Formula 2 is plottedon the Y axis, the value of K_(D) for adenosine was determined by theleast squares method using the Solver function of Office Excel 2007(Microsoft).

R=Rmax×conc/(K _(D)+conc)  (Formula 2)

In Formula 2, conc represents adenosine concentration (mol/l) while Rmaxrepresents the value of response expected for the maximal binding ofadenosine to antibody. Measured response values were extracted by usingScrubber2 (BioLogics. Inc).

The KD of D12 determined by the measurement described above was 8.5μmol/l for ATP, 0.25 μmol/l for ADP, or 1100 μmol/l for adenosine. Thisresult demonstrates that D12 has binding activity to ATP, ADP, andadenosine; and it also suggests that D12 has binding activity to AMP(adenosine monophosphate) and cAMP (cyclic adenosine monophosphate).

Example 8 Design of Library Using Anti-ATP/Adenosine Antibodies toPrepare ATP/Adenosine Switch Antibodies

In cancer tissues and inflamed tissues, not only the adenosine but alsothe ATP concentration is known to be high. Thus, it is beneficial to useantibodies for which both adenosine and ATP (referred to asATP/adenosine in this Example) can serve as a switch (specifically,antibodies that can bind to antigens when adenosine or ATP is present ata high concentration) as well as antibodies for which either adenosineor ATP alone serves as a switch. ATNLSA1-4_D12 described in Example 7-4is an antibody that binds to ATP/adenosine. As shown in FIG. 19,ATP/adenosine is thought to be fit between the antibody and its targetantigen, and thus the antibody comprises an antibody variable regionthat comes in contact with the target antigen. Thus, the presentinventors conceived that synthetic antibody libraries that can isolateATP/adenosine switch antibodies whose binding activity to arbitraryantigens is altered depending on the presence of ATP/adenosine could beconstructed by collecting, as a library, antibody variable regionsegments that are capable of establishing contact with a target antigenand maintaining ATP/adenosine binding.

The crystal structure of the complex between ATP and ATP/adenosineantibody ATNLSA1-4_D12 obtained from a human antibody library asdescribed in Example 7-4 was analyzed. The result of crystal structureanalysis revealed the mode of adenosine (or ATP) recognition by theantibody as well as identification of amino acid residues that areconsidered not to be substantially involved in adenosine (or ATP)binding in the antibody variable region. Amino acid residues that havebeen identified to be closely involved in the adenosine (ATP) bindingare Ser52, Ser52a, Arg53, Gly96, Leu100a, and Trp100c (Kabat numbering)in the heavy chain.

In designing such a library, positions that meet at least one of theconditions described below were selected as suitable for the libraryconstruction.

Condition 1: a position that is not closely involved in ATP binding, orif involved in the binding, a position having an amino acid other thanthe wild-type sequence that does not inhibit the ATP binding;

Condition 2: a position that is polymorphic to a certain extent in termsof amino acid occurrence frequency; and

Condition 3: a position that is not essential for the formation ofcanonical structure.

In regions contained in both heavy chain and light chain of theATNLSA1-4_D12 sequence and that meet the conditions described above,amino acids in the CDR1 and CDR2 regions that have an occurrencefrequency of 2% or more in the germ line, as well as amino acids in theCDR3 region that have an occurrence frequency of 1% or more in the germline were comprehensively substituted. These substitutions were combinedto construct multiple variants of ATNLSA1-4_D12.

Alteration sites in the heavy chain (in the Table, positions indicatedby “Kabat” according to Kabat numbering), as well as amino acids beforealteration (in the table, amino acids referred to as “natural sequence”)at the sites and amino acids after alteration (in the table, amino acidsreferred to as “altered amino acids”) are shown in Table 9.

TABLE 9 HCDR1 HCDR2 HCDR3 Kabat 31 32 35 55 57 58 96 97 99 100 100a Wildtype sequence T Y N N I N G R G D L Altered A A A A A A A A A amino Cacid E E D D D D D D D G G G G F F F F I I H H H H K K K K K M M L L N NN N N N N Q P S S S S S S S S S S R R R R R T T T T T W W V V V V V Y YY Y Y Y Y

Alteration sites in the light chain (in the Table, positions indicatedby “Kabat” according to Kabat numbering), as well as amino acids beforealteration (in the table, amino acids referred to as “natural sequence”)at the sites and amino acids after alteration (in the table, amino acidsreferred to as “altered amino acids”) are shown in Table 10.

TABLE 10 LCDR1 LCDR2 LCDR3 Kabat 26 27 27a 27b 27c 28 29 31 32 50 51 5253 54 55 89 90 91 92 93 94 95a 96 97 Wild type sequence T S S D V G G NY E V S K R P S S Y A G S N V V Altered A A A A A A A A A A A A A aminoC acid E E E E E D D D D D D D D D D D D D D D G G G G G G G G G F F F FF I I I I I I H H H H H K K K K K K K K M M L L L L L L L N N N N N N NN N N N N Q Q Q Q Q Q P P P S S S S S S S S S S S S R R R R R R R R T TT T T T T T T T T T T T T W W W V V V V V V Y Y Y Y Y Y Y Y Y

Each variant expressed and purified by the method described in Example7-1 was assayed for its ATP and adenosine binding by the same method asdescribed in Example 7-6 using Biacore. Based on the assay result, theaffinity of each variant for ATP was calculated as a KD value. Sites inthe heavy chain, where alteration does not reduce the ATP-bindingability to less than ⅕ of the binding ability of ATNLSA1-4_D12(specifically, where the KD value is lower than 42.5 μmol/l), and sitesin the light chain where the ATP-binding ability is larger than that ofATNLSA1-4_D12 (specifically, where the KD value is smaller than 8.5μmol/l), were assessed to be plausible for alteration. Amino acidssubstituted at those sites were judged to be appropriate for inclusionin the library (flexible residues to be introduced into library).

Based on the assessment result on the ATP-binding ability of eachvariant, the ATP-binding ability was predicted to be reduced bycollecting each site to construct a library. Thus, substitutions wereintroduced at sites close to positions that are expected to be involvedin ATP binding, and various variants resulting from combination of thesesubstitutions were comprehensively assessed to test whether it ispossible to identify alterations which are expected to have effect ofaugmenting the ATP-binding ability. Such alteration sites (positionsindicated by “Kabat” according to Kabat numbering in the Table), andamino acids before alteration (amino acids referred to as “wild typesequence” in the table) and amino acids after alteration (amino acidsreferred to as “altered amino acids” in the table) at the sites areshown in Table 11.

TABLE 11 HCDR1 HCDR2 HCDR3 LCDR3 Kabat 33 50 56 95 98 100b 95 WiLd typesequence T S Y F K N N Altered A A A A A A A A amino C acid E E E E E EE D D D D D D D G G G G G G G F F F F F F F I I I I I I I I H H H H H HH K K K K K K K M M M M M M M M L L L L L L L L N N N N Q Q Q Q Q Q Q PP P P P P P S S S S S S R R R R R R R T T T T T T W W W W W W W W V V VV V V V V Y Y Y Y Y Y Y

Each variant expressed and purified by the method described in Example7-1 was assayed for its ATP and adenosine binding by the same assaymethod using Biacore as described in Example 7-6. The assay resultshowed that the ATP and adenosine binding was expected to be augmentedby alterations at positions 56 and 100 and such according to Kabatnumbering (for example, amino acid alteration such as Tyr56His andAsn100bLeu). It was determined that amino acids substituted at the sitescould be included in a library (flexible residues to be introduced intoa library).

In the CDR regions of ATNLSA1-4_D12, amino acid repertoires containingamino acids selected by the above-described variant analysis as suitableto be included in a library (flexible amino acid residues to beintroduced into a library) and amino acids before alteration of theamino acids (specifically, amino acids included in the natural sequenceof ATNLSA1-4_D12), and sites comprising such repertoires were designedto construct a library for preparation of ATP/adenosine switchantibodies. The library was constructed in such a manner that in anamino acid repertoire the amino acid occurrence frequency is the samefor every amino acid (for example, when there are ten types of aminoacids in an amino acid repertoire, each amino acid occurs at 10%,).

Sites comprising amino acid repertoires in the heavy chain (positionsindicated by “Kabat” according to Kabat numbering in the Table) andamino acid repertoires at the sites are shown in Table 12. Sitescomprising amino acid repertoires in the light chain (positionsindicated by “Kabat” according to Kabat numbering in the Table) andamino acid repertoires at the sites are shown in Table 13.

TABLE 12 HCDR1 HCDR2 HCDR3 Kabat 31 32 35 55 56 57 58 59 95 97 98 99 100100a 100b Wild type sequence T Y N N Y I N Y F R K G D L N Altered A 17%25% 11% 5% amino C acid E 9% 5% D 13% 9% 5% G 33% 17% 13% 11% 9% 5% F33% 13% 50% 9% 5% I 25% 11% 5% H 33% 50% 50% 13% 11% 9% 5% 17% K 25% 11%33% 9% 5% M 11% 5% 17% L 50% 11% 33% 5% 17% 50% N 50% 17% 13% 9% 5% 50%Q 9% 5% P 5% S 33% 17% 13% 5% R 17% 25% 11% 33% 9% 5% 17% T 33% 17% 13%5% W 9% 5% 17% V 11% 5% Y 33% 50% 13% 50% 50% 9% 5% 17%

TABLE 13 LCDR1 LCDR2 LCDR3 Kabat 27a 29 50 51 54 90 91 92 93 94 95 95a96 97 Wild type sequence S S E V R S Y A G S N N V V Altered A 17% 17%14% 13% 6% 17% amino C acid E 14% 17% 6% D 17% 14% 14% 13% 6% 11% G 17%14% 25% 14% 13% 6% 11% 17% F 17% 6% I 14% 13% 6% 11% H 6% 11% K 14% 50%14% 6% M 6% 17% L 25% 6% 11% 33% 17% N 25% 14% 13% 6% 11% Q 14% 6% 11% P6% 33% S 50% 17% 14% 25% 17% 14% 13% 6% 11% 17% R 50% 14% 17% 13% 6% T50% 17% 25% 25% 14% 17% 14% 13% 6% W 14% 6% V 25% 25% 14% 33% 17% Y 14%14% 17% 14% 6% 11%

The result of sequence analysis suggests that the framework ofATNLSA1-4_D12 was derived from germ line VH3-21. Then, for the purposeof improving antibody stability, the framework sequence of ATNLSA1-4_D12was restored to the germ line sequence VH3-21 by introducing into theframework sequence of ATNLSA1-4_D12, alterations Gln01Glu, Gln05Val,Asp10Gly, Asn30Ser, Leu48Val, and Asn58Tyr (numerals represent Kabatnumbers). ATNLSA1-4_D12 variants expressed and purified by the methoddescribed in Example 7-1 were measured for their Tm by DSC. DSCmeasurement was carried out by a method known to those skilled in theart. Tm of the variant which results from adding these alterations toATNLSA1-4_D12 was markedly improved from 74.37° C. to 81.44° C., andstabilization of the structure was observed. It is sometimes preferableto use highly stable frameworks for antibody libraries, and thus aframework sequence to which alterations described above had been addedwas used as the framework sequence of a library. The framework used forthe library is shown in Table 14.

TABLE 14 Framework SEQ ID NO: Sequence Heavy chain framework 1 56EVQLVESGGGLVKPGGFLRLSCAASGFTFS Heavy chain framework 2 57 WVRQAPGKGLEWVSHeavy chain framework 3 58 RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHeavy chain framework 4 59 WGQGTLVTVSS Light chain framework 1 60QSALTQPPSASGSPGQTVTISC Light chain framework 2 61 SWYQQHPGKAPKLMIYLight chain framework 3 62 GVPDRFSGSKSGNTASLTVSGLQAEDEADYFCLight chain framework 4 63 FGGGTKLTVL

Genes were synthesized to comprise respective sequences in a librarydesigned as described above (DNA2.0), and the gene library was amplifiedwith primers that are capable of amplifying VH and VL respectively, byusing a collection (library) of the respective genes as a template. Thesequences of primers used for VL amplification are shown in SEQ ID NOs:102 and 103, while the sequences of primers used for VH amplificationare shown in SEQ ID NOs: 104 and 105. The amplified rationally designedgene library of the heavy-chain and light-chain variable regions ofhuman antibody was inserted into an appropriate phagemid vector carryingboth a human IgG-derived CH1 sequence and a human IgG-derived lightchain constant region sequence. The phagemid vector was introduced intoE. coli by electroporation to construct a rationally designed librarywhich presents Fab domains containing a human antibody variableregion-constant region, and from which one can isolate antibodies thatare capable of binding to antigens via adenosine or ATP as a switch.Such a rationally designed library which is constituted with various Hchains and L chains that have adenosine- or ATP-binding activity isexpected to be useful as a library containing human antibodies that,with the adenosine (or ATP) is fit in between antibody and antigen asshown in FIG. 19, can efficiently obtain adenosine/ATP switch antibodiesagainst any arbitrary antigen. Furthermore, as described above, sinceATNLSA1-4_D12 binds not only to adenosine and ATP but also to ADP, itwas predicted to have binding activity to AMP and cAMP which arestructurally similar to ATP, ADP, and adenosine. This suggests that suchlibraries are useful for isolating switch antibodies whose bindingactivity to arbitrary target antigens is altered depending on thepresence of any one or more small molecules of ATP, ADP, AMP, cAMP, andadenosine.

Example 9 Construction of an Immune Library to ObtainAdenosine/ATP/Adenosine Switch Antibodies Containing Anti-ATP/AdenosineAntibody Repertoires

Multiple phage-display libraries of rabbit antibodies which present Fabdomains comprising rabbit antibody sequences were constructed using as atemplate mRNA collected from a B cell population selected using MACS andFACS as described in Example 5-4, which expressesadenosine-PEG-biotin-binding antibodies. The construction method wascarried out by referring to Rader (Methods Mol. Biol. (2009) 525,101-28).

More specifically, cDNA was prepared by reverse transcription using as atemplate mRNA collected from 600,000 cells of the above-described Bcells which were selected from nine immunized rabbits. Using the cDNA asa template, the heavy chain variable region sequence and the light chainvariable region-constant region sequence were amplified by PCR using theprimers shown in Table 15 under adequate conditions.

TABLE 15 SEQ Primer name ID NO: Sequence primer 1 64TATTACTCGCGGCCCAGCCGGCCATGGCAGCC WTCGANWTGACCCAGACT primer 2 65TATTACTCGCGGCCCAGCCGGCCATGGCAGCC TATGATNTGACCCAGACT primer 3 66TATTACTCGCGGCCCAGCCGGCCATGGCAGCB CAAGTGCTGACCCAGACT primer 4 67TATTACTCGCGGCCCAGCCGGCCATGGCAGCC MTYGTGATGACCCAGACT primer 5 68TATTACTCGCGGCCCAGCCGGCCATGGCAGCC GCCGTGCTGACCCAGACT primer 6 69TATTACTCGCGGCCCAGCCGGCCATGGCGGCT GACATTGTGATGACCCAG primer 7 70TATTACTCGCGGCCCAGCCGGCCATGGCCGCC GAYRTYGTGATGACCCAG primer 8 71CTCTTCTAGAACGCGTCTAAGCGTCACCCCTA TTGAAGCTC primer 9 72TATTACTCGCGGCCCAGCCGGCCATGGCGCAG CYYGTGCTGACTCAGTCGCCCTC primer 10 73CTCTTCTAGAACGCGTCTAAGCTTCTGCAGGG GCCAGGCTCTTC primer 11 74TTCCGCCTCGGCGCTAGCCCAGGAGCAGSTGG WGGAGTCC primer 12 75TTCCGCCTCGGCGCTAGCCCAGTCNNTGGAGG AGTCCGGG primer 13 76TTCCGCCTCGGCGCTAGCCCAGTCGNNGGAGG AGTCCGGG primer 14 77TTCCGCCTCGGCGCTAGCCCAGCAGCAGCTGG WGGAGTCC

A combination of an amplified library of rabbit antibody heavy chainvariable region genes and a library of rabbit antibody light chainvariable region-constant region genes was inserted into an appropriatephagemid vector carrying a rabbit IgG-derived CH1 sequence. The phagemidvector was introduced into E. coli by electroporation to construct aphage-display library of rabbit antibodies (hereinafter, an antibodylibrary from adenosine-immunized rabbits) which presents Fab domainscontaining a rabbit antibody variable region-constant region, and fromwhich one can isolate antibodies that are capable of binding to antigensvia adenosine or ATP as a switch. Such an adenosine immune library whichis constituted by various H chains and L chains that exhibit adenosinebinding property is expected to be useful as an immune library, withadenosine (or ATP) is sandwiched in between antibody and antigen asshown in FIG. 20, that can isolate adenosine/ATP switch antibodiesagainst any arbitrary antigen.

Example 10 Acquisition of Antibodies that Bind to Antigens in thePresence of Adenosine and ATP from Antibody Library Using Phage DisplayTechniques

(10-1) Acquisition of Antibodies that Bind to Antigens in the Presenceof Small Molecules from Library Using a Mixture of Adenosine and ATP

Antibodies that exhibit antigen-binding activity in the presence ofadenosine and/or ATP were obtained from the constructed phage-displaylibrary of antibodies from adenosine-immunized rabbits and thephage-display library of rationally designed antibodies. To obtainantibodies, phages displaying antibodies that exhibit the ability tobind to antigens captured by beads in the presence of adenosine and ATPwere collected, and then the phages were collected in eluate from thebeads in the absence of adenosine and ATP.

Phages were produced in E. coli containing the phagemid vectorconstructed for phage display. To the culture medium of E. coli in whichphage production was carried out, 2.5 M NaCl/10% PEG was added toprecipitate phages. The precipitated phage fraction was diluted with TBSto prepare a library suspension. Then, BSA was added at a finalconcentration of 4% to the phage library suspension. Panning wasperformed using antigen-immobilized magnetic beads. The magnetic beadsused were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) and Streptavidin coated beads (Dynabeads M-280Streptavidin).

500 pmol of biotin-labeled antigen and a final concentration of 1 mMATP-Na and adenosine were each added to the prepared phage librarysuspension. The phage library suspension was contacted with the antigen,adenosine, and ATP at room temperature for 60 minutes. The BSA-blockedmagnetic beads were added to the phage library suspension, and theantigen-phage complex was allowed to bind to the magnetic beads at roomtemperature for 15 minutes. The beads were washed once with ATP- andadenosine-dissolved TBS. Then, the beads were combined with 0.5 ml of 1mg/ml trypsin. Immediately after the beads at room temperature weresuspended for 15 minutes, a phage suspension was collected from theisolated beads using a magnetic stand. The collected phage suspensionwas added to 10 ml of E. coli cells of strain ER2738 at the logarithmicgrowth phase (OD600=0.4 to 0.7). The E. coli was incubated at 37° C.with gentle stirring for one hour to be infected by phage. The infectedE. coli was seeded in a 225 mm×225 mm plate. Then, phages were collectedfrom the culture medium of the seeded E. coli to prepare a liquid stockof phage library.

The first round of panning was carried out to collect phages that arecapable of antigen binding in the presence of adenosine and ATP, whilethe second and subsequent rounds of panning were performed to enrichphages that are capable of antigen binding only in the presence ofadenosine and ATP. Specifically, 40 pmol of biotin-labeled antigen and afinal concentration of 1 mM adenosine and ATP were each added to theprepared phage library suspension. Thus, the phage library was contactedwith antigen, adenosine, and ATP for 60 minutes at room temperature.BSA-blocked magnetic beads were added, and the antigen-phage complex wasallowed to bind to the magnetic beads for 15 minutes at roomtemperature. The beads were washed with 1 ml of adenosine andATP-dissolved TBST (hereinafter referred to as (adenosine+ATP)/TBST),adenosine, and adenosine and ATP-dissolved TBS (hereinafter referred toas (adenosine+ATP)/TBS). Then, the beads were combined with 0.5 ml ofTBS. Immediately after the beads were suspended at room temperature, aphage suspension was collected from the isolated beads using a magneticstand. After this treatment was repeated, the two separately elutedphage suspensions were combined together. The pIII protein (helperphage-derived protein pIII) that does not display Fab was cleaved offfrom phages by adding 5 μl of 100 mg/ml trypsin to the collected phagesuspension to eliminate the ability of phages that do not display Fab toinfect E. coli. The phages collected from the trypsinized phagesuspension were added to 10 ml of E. coli cells of strain ER2738 at thelogarithmic growth phase (OD600=0.4 to 0.7). The E. coli was incubatedat 37° C. with gentle stirring for one hour to be infected by phage. Theinfected E. coli was seeded in a 225 mm×225 mm plate. Then, phages werecollected from the culture medium of the seeded E. coli to prepare aphage library suspension. Panning was performed three times to isolateantibodies that have antigen-binding activity in the presence ofadenosine and ATP.

(10-2) Acquisition of Antibodies that Bind to Antigens in the Presenceof Adenosine and ATP from Antibody Library Using a Negative SelectionMethod

A phage-display library of antibodies constructed from rabbits immunizedwith adenosine or a phage-display library of rationally designedantibodies was screened for antibodies that exhibit antigen-bindingactivity in the presence of adenosine and/or ATP. As a first step ofscreening, the phage-display antibody library was contacted withbiotin-labeled antigen-streptavidin in the absence of adenosine and ATPto eliminate phages displaying antibodies that have antigen-bindingactivity even in the absence of adenosine and ATP. Then, panning wasperformed in the same manner in the presence of adenosine and ATP toscreen for antibodies that exhibit antigen-binding activity in thepresence of adenosine and ATP.

Phages were produced in E. coli containing the constructed phage-displayphagemid. To the culture medium of E. coli in which phage productiontook place, 2.5 M NaCl/10% PEG was added to precipitate phages. Theprecipitated phage fraction was diluted with TBS to prepare a librarysuspension. Then, BSA was added at a final concentration of 4% to thephage library suspension. Panning was performed usingantigen-immobilized magnetic beads. The magnetic beads used wereNeutrAvidin coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) andStreptavidin coated beads (Dynabeads M-280 Streptavidin).

Together with 250 pmol of biotin-labeled antigen, a mixture of adenosineand ATP was added at a final concentration of 1 mM to the prepared phagelibrary suspension. Thus, the phage library suspension was contactedwith the antigen, adenosine, and ATP for 60 minutes at room temperature.Then, BSA-blocked magnetic beads were added to the phage librarysuspension, and allowed to bind to the antigen-phage complex at roomtemperature for 15 minutes. The beads were washed once with(adenosine+ATP)/TBS. Then, the beads were combined with 0.5 ml of 1mg/ml trypsin solution. Immediately after the beads were suspended atroom temperature for 15 minutes, a phage suspension was collected fromthe isolated beads using a magnetic stand. The collected phagesuspension was added to 10 ml of E. coli cells of strain ER2738 at thelogarithmic growth phase (OD600=0.4 to 0.7). The E. coli was incubatedat 37° C. with gentle stirring for one hour to be infected by phage. Theinfected E. coli was seeded in a 225 mm×225 mm plate. Then, phages werecollected from the culture medium of the seeded E. coli to prepare aliquid stock of phage library.

The first round of panning was carried out to collect phages that arecapable of binding in the presence of adenosine and ATP, while thesecond and subsequent rounds of panning were performed to enrich phagesthat are capable of antigen binding only in the presence of adenosineand ATP. Specifically, 250 pmol of biotinylated antigen was added toBSA-blocked Sera-Mag NeutrAvidin beads, and allowed to bind at roomtemperature for 15 minutes. The beads were washed three times with TBS.The phage library suspension subjected to BSA blocking was added to thewashed beads, and allowed to bind at room temperature for one hour.Phages that did not bind to the antigens or beads were collected byisolating the beads using a magnetic stand. Forty pmol of biotin-labeledantigen, and a final concentration of 1 mM adenosine and ATP were eachadded to the collected phages. Thus, the phage library was contactedwith the antigen, adenosine, and ATP for 60 minutes at room temperature.Then, BSA-blocked magnetic beads were added to the mixture of thelabeled antigen, adenosine, ATP, and phage library, and allowed to bindto the antigen-phage complex for 15 minutes at room temperature. Thebeads were washed with 1 ml of (adenosine+ATP)/TBST and(adenosine+ATP)/TBS. Then, 0.5 ml of 1 mg/ml trypsin solution was addedto the mixture. After the mixed suspension was stirred at roomtemperature for 20 minutes, phages were collected from the beads thathad been separated using a magnetic stand. The collected phages wereadded to 10 ml of E. coli cells of strain ER2738 at the logarithmicgrowth phase (OD600=0.4 to 0.7). The E. coli was incubated at 37° C.with gentle stirring for one hour to be infected by phage. The infectedE. coli was seeded in a 225 mm×225 mm plate. Panning was performed threetimes to isolate antibodies that have antigen-binding activity in thepresence of adenosine and ATP.

(10-3) Acquisition of Antibodies that Bind to Antigens in the Presenceof Adenosine and ATP from Antibody Library Using an Alternating PanningMethod

A phage-display antibody library constructed from rabbits immunized withadenosine or a phage-display library of rationally designed antibodiesis screened for antibodies that exhibit antigen-binding activity in thepresence of adenosine and/or ATP. As a first step of screening, thephage-display antibody library is contacted with biotinylated adenosineand ATP-NeutrAvidin in the presence of non-labeled antigens to collect aphage-display library of antibodies that bind to adenosine and/or ATP inthe presence of the antigen. Then, the phage-display antibody library iscontacted with biotinylated antigen-streptavidin in the presence ofadenosine and ATP to collect antibodies that bind to the antigen in thepresence of adenosine and ATP. Thus, screening is carried out forantibodies that have antigen-binding activity in the presence ofadenosine and ATP by performing panning in the alternating mannerdescribed above.

Phages are produced in E. coli containing the phagemid vectorconstructed for phage display. To the culture medium of E. coli in whichphage production is took place, 2.5 M NaCl/10% PEG is added toprecipitate phages. The precipitated phage fraction is diluted with TBSto prepare a library suspension. Then, BSA is added at a finalconcentration of 4% to the phage library suspension. Panning isperformed using antigen-immobilized magnetic beads. The magnetic beadsused are NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) and Streptavidin coated beads (Dynabeads M-280Streptavidin).

Together with 1000 pmol of non-labeled antigen, 250 pmol of biotinylatedATP, 2′-Adenosine-PEG-Biotin, and 5′-Adenosine-PEG-Biotin are added tothe prepared phage library suspension. Thus, the phage librarysuspension is contacted with the antigen, adenosine, and ATP at roomtemperature for 60 minutes. Then, BSA-blocked magnetic beads are addedto the phage library suspension, and the complex of phage with theantigen, and adenosine and/or ATP is allowed to bind to the magneticbeads at room temperature for 15 minutes. The beads are washed once withTBS containing 1000 pmol of the antigen. Then, the beads are combinedwith 0.5 ml of 1 mg/ml trypsin solution. Immediately after the beads aresuspended at room temperature for 15 minutes, a phage suspension iscollected from the isolated beads using a magnetic stand. The collectedphage suspension is added to 10 ml of E. coli cells of strain ER2738 atthe logarithmic growth phase (OD600=0.4 to 0.7). The E. coli isincubated at 37° C. with gentle stirring for one hour to be infected byphage. The infected E. coli are plated onto a 225 mm×225 mm plate. Then,phages are collected from the culture medium of the seeded E. coli toprepare a phage library suspension.

A second round of panning is performed to enrich phages that are capableof binding to the biotinylated antigen in the presence of adenosine andATP. Specifically, 40 pmol of biotinylated antigen and a finalconcentration of 1 mM adenosine and ATP are added to the prepared phagelibrary solution. Thus, the phage library suspension is contacted withthe antigen, as well as adenosine and ATP for 60 minutes at roomtemperature. Then, BSA-blocked magnetic beads are added to the phagelibrary solution, and the complex of phage with the antigen as well asadenosine and/or ATP is allowed to bind to the magnetic beads at roomtemperature for 15 minutes. The beads are washed three times with TBSTcontaining adenosine and ATP at a final concentration of 1 mM, and twicewith TBS containing adenosine and ATP at a final concentration of 1 mM.Then, the beads are combined with 0.5 ml of 1 mg/ml trypsin solution.Immediately after the beads are suspended at room temperature for 15minutes, a phage solution is collected from the isolated beads using amagnetic stand. The collected phage solution is added to 10 ml of E.coli cells of strain ER2738 at the logarithmic growth phase (OD600=0.4to 0.7). The E. coli is incubated at 37° C. with gentle stirring for onehour to be infected by phage. The infected E. coli is seeded in a 225mm×225 mm plate. Then, phages are collected from the culture medium ofthe seeded E. coli to prepare a phage library solution.

At subsequent even-numbered rounds, panning is performed in the samemanner as the second-round panning. However, in the fourth andsubsequent rounds of panning, the number of bead washes with(adenosine+ATP)/TBST and (adenosine+ATP)/TBS are both increased to fivetimes.

A third round of panning is performed to also enrich phages that arecapable of binding to biotinylated adenosine and ATP in the presence ofthe antigen. Specifically, together with 250 pmol of biotinylated ATP,2′-adenosine-PEG-Biotin and 5′-adenosine-PEG-Biotin, 1000 pmol ofnon-labeled antigen is added to the prepared phage library solution.Thus, the phage library solution is contacted with the antigen, as wellas adenosine and ATP for 60 minutes at room temperature. Then,BSA-blocked magnetic beads are added to the phage library solution, andthe complex of phage with the antigen as well as adenosine and/or ATP isallowed to bind to the magnetic beads at room temperature for 15minutes. The beads are washed three times with TBST containing 1000 pmolof the antigen, and twice with TBS containing 1000 pmol of the antigen.Then, the beads are combined with 0.5 ml of a 1 mg/ml trypsin solution.Immediately after the beads are suspended at room temperature for 15minutes, a phage solution is collected from the isolated beads using amagnetic stand. The collected phage solution is added to 10 ml of E.coli cells of strain ER2738 at the logarithmic growth phase (OD600=0.4to 0.7). The E. coli is incubated at 37° C. with gentle stirring for onehour to be infected by phage. The infected E. coli is seeded in a 225mm×225 mm plate. Then, phages are collected from the culture medium ofthe seeded E. coli to prepare a phage library solution.

At subsequent odd-numbered rounds, panning is performed in the samemanner as the third-round panning. However, in the fourth and subsequentrounds of panning, the number of bead washes with TBST containing theantigen and TBS containing the antigen is both increased to five times.Alternatively, regardless of whether it is an odd- or even-numberedround, panning in the third and subsequent rounds is always performed inthe same manner as the third-round panning. However, in the fourth andsubsequent rounds of panning, the number of bead washes with TBSTcontaining antigen and TBS containing antigen is both increased to fivetimes.

(10-4) Assessment of Binding Activity in the Presence or Absence ofAdenosine and/or ATP by Phage ELISA

Phage-containing culture supernatants were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained by the method described above. Thecollected culture supernatants were treated by ultrafiltration usingNucleoFast 96 (MACHERY-NAGEL). 100 μl of the culture supernatants wereadded to each well of NucleoFast 96, and it was centrifuged (4500 g for45 minutes) to remove flow through. After addition of 100 μl of H₂O, theNucleoFast 96 was washed by centrifugation (4500 g for 30 minutes).Finally, 100 μl of TBS was added, and the NucleoFast 96 was allowed tostand for five minutes at room temperature. A phage suspension wascollected from the supernatant in each well of the NucleoFast 96.

The purified phages, to which TBS or (adenosine+ATP)/TBS was added, weresubjected to ELISA by the following procedure. A StreptaWell 96microtiter plate (Roche) was coated overnight with 100 μl of TBScontaining biotin-labeled antigen. After the antigen was removed bywashing each well of the plate with TBST, the wells were blocked with250 μl of 2% skim milk/TBS for one hour or more. 2% skim milk/TBS wasremoved, and then the prepared, purified phages were added to each well.The plate was allowed to stand at 37° C. for one hour to allow bindingof antibody-displaying phages to the antigen in each well in thepresence or absence of adenosine and/or ATP. After washing with TBST or(adenosine+ATP)/TBST, HRP-conjugated anti-M13 antibody (AmershamPharmacia Biotech) diluted with TBS or (adenosine+ATP)/TBS was added toeach well. The plate was incubated for one hour. Following washes withTBST or (adenosine+ATP)/TBST, the TMB single solution (ZYMED) was addedto each well. The chromogenic reaction in the solution of each well wasterminated by adding sulfuric acid. Then, the developed color wasassessed by measuring absorbance at 450 nm. The result revealed that forthree types of antigens: human IL6, human IL6 receptor, and HAS (humanserum albumin), there were multiple types of antibodies bound in thepresence of small molecules. Antibodies that bind in the presence of ATPwere also obtained from the human naïve antibody library. Meanwhile,switch antibodies to human IL6, human IL6 receptor, and HAS could beobtained with greater efficiency. The result of phage ELISA is shown inTable 16.

TABLE 16 human IL6 human IL6R HSA Number of panning  4  3  4  4 Numberof clones subjected 96 96 96 96 to ELISA Number of positive clones 35 2364 52 (S/N ratio > 10) Nutter of dependent clones 18 22 64 50 (SM +/−ratio > 2) Number of dependent clone  2 17 35  5 sequences

(10-5) Assessment for Binding Ability of Switch Antibodies WhoseAntigen-Binding Activity is Altered Depending on the Presence ofAdenosine and ATP, and Sequence Analysis

Genes were amplified using specific primers (SEQ ID NOs: 111 and 112)from clones that had been assessed to have antigen-binding activity inthe presence of adenosine or ATP based on the phage ELISA resultdescribed in (10-4). The nucleotide sequences of the genes wereanalyzed, and the result showed that multiple antibodies that bind toantigens, human IL6, HSA, and human IL6R, and have sequences differentfrom one another were obtained. The amino acid sequences ofI6DL2C1-4_(—)076 (antibody to human IL6), HSDL3C5-4_(—)015 (antibody toHSA), and 6RAD2C1-4_(—)011 and 6RAD2C1-4_(—)076 (antibodies to humanIL-6R) are shown in Table 17.

TABLE 17 Heavy chain Light chain Clone name SEQ ID NO SEQ ID NOI6DL2C5-4_076 SEQ ID NO: 78 SEQ ID NO: 79 HSDL3C5-4_015 SEQ ID NO: 80SEQ ID NO: 81 6RAD2C1-4_011 SEQ ID NO: 82 SEQ ID NO: 83 6RAD2C1-4_076SEQ ID NO: 84 SEQ ID NO: 85

(10-6) Identification of Small Molecules Required for Antigen Binding ofthe Obtained Antibodies

Each of the obtained antibodies I6DL2C1-4_(—)076, HSDL3C5-4_(—)015,6RAD2C1-4_(—)011, and 6RAD2C1-4_(—)076 was subjected to ELISA. The smallmolecules used were 1 mM ATP, adenosine, and a mixture thereof. Theantigens used were biotin-labeled human IL6, human IL6R, and HSA.

First, a StreptaWell 96 microtiter plate (Roche) was coated at roomtemperature for one hour or more with 100 μl of TBS containing abiotin-labeled antigen. Following TBST wash to remove the unboundbiotin-labeled antigen from the plate, each well was blocked with 250 μlof 2% skim milk/TBS for one hour or more. After 2% skim milk/TBS wasremoved from each well, 50 μl of the antibody-displaying phage was addedto the plate. The plate was allowed to stand at room temperature for onehour to allow binding of each phage to the biotin-labeled antigen ineach well in the presence or absence of ATP and/or adenosine. Afterwashing with TBST with or without ATP and/or adenosine, anHRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) dilutedwith TBS or (adenosine and/or ATP)/TBS was added to each well. The platewas incubated for one hour. Following wash with TBST with or withouteach small molecule, the TMB single solution (ZYMED) was added to eachwell. The chromogenic reaction in the solution of each well wasterminated by adding sulfuric acid. Then, the developed color wasassessed by measuring absorbance at 450 nm (FIGS. 25, 26, and 27).

Example 11 Binding Activity of Antibodies Obtained as Described inExample 2 to Human IL-6 in the Presence of Amino Acid Metabolites Otherthan Kynurenine

Antibody I6NMSC1-3_A11 obtained as described in Example 2-4, which bindsto human IL-6 in the presence of small molecules, is an antibody thatbinds to human IL-6 in the presence of kynurenine as described inExample 3-2. Kynurenine is a tryptophan metabolite, which is convertedto anthranilic acid by kynureninase; to 3-hydroxykynurenine bykynurenine 3-hydroxylase; and to kynurenic acid by kynurenineaminotransferase (Stefan Lob et. al., Nat Rev Cancer. (2009) 9 (6),445-452). Amino acid metabolites such as tryptophan metabolites wereassessed as to whether they are appropriate as a non-limiting embodimentof cancer tissue-specific compounds of the present invention,particularly cancer cells-specific metabolites of the present invention.

I6NMSC1-3_A11 described in Example 3-2 which has antigen-bindingactivity in the presence of kynurenine, a known anti-human IL-6 antibodyCLB8-F1, and GC413 as a negative control were subjected to ELISA underthe seven conditions described Table 18. Meanwhile, each amino acid andmetabolites thereof were appropriately prepared at the concentrationsshown in Table 18 using the buffers indicated in Table 4. The antigenused was biotin-labeled human IL-6.

TABLE 18 Condition Small molecule Concentration 1 Kynurenine 100 μM 2Tryptophan 100 μM 3 Phenylalanine 100 μM 4 Anthranilic acid 100 μM 53-Hydroxykynurenine 100 μM 6 Kynurenic acid 100 μM 7 — —

First, a StreptaWell 96 microtiter plate (Roche) was coated at roomtemperature for one hour or more with 100 μl of PBS containing abiotin-labeled antigen. After washing with Wash buffer to remove theunbound antigen from the plate, each well was blocked for one hour ormore with 250 μl of Blocking Buffer. After Blocking Buffer was removedfrom each well, the purified IgGs were prepared to 2.5 μg/ml in SampleBuffer containing small molecules at the final concentrations shown inTable 18, and each was aliquoted at 100 μl into the plate. The plate wasallowed to stand at room temperature for one hour to allow binding ofeach IgG to the antigen in each well. After washing with Wash Buffercontaining amino acids or amino acid metabolites at the finalconcentrations shown in Table 18, an HRP-conjugated anti-human IgGantibody (BIOSOURCE) diluted with Sample Buffer containing the sameamino acids and amino acid metabolites was added to each well. The platewas incubated for one hour. Following wash with Wash Buffer containingeach amino acid or amino acid metabolite, the TMB single solution(ZYMED) was added to each well. The chromogenic reaction in the solutionof each well was terminated by adding sulfuric acid. Then, the developedcolor was assessed by measuring absorbance at 450 nm. The compositionsof buffers used are shown in Table 4.

The measurement result is shown in FIG. 21. The absorbance for CLB8-F1was constant regardless of the type and presence of small molecules.Meanwhile, the absorbance for I6NMSC1-3_A11 was markedly lower undercondition 7 (in the absence of small molecules) as compared to undercondition 1 (kynurenine solution). Furthermore, the absorbance forI6NMSC1-3_A11 was high under condition 2 (tryptophan solution) andcondition 5 (3-hydroxykynurenine solution) as well as under condition 1.This shows that I6NMSC1-3_A11 is an antibody that binds to human IL-6 asan antigen not only in the presence of kynurenine, but also in thepresence of an amino acid (tryptophan) as a kynurenine precursor or inthe presence of a kynurenine metabolite.

This finding suggests that the same method can be used to obtainantibodies that bind to an antigen of interest not only in the presenceof a single type of amino acid metabolite but also in the presence ofmultiple different types of amino acids or amino acid metabolites.

Example 12 Acquisition of Antibodies that Bind to Human IL-6 in thePresence of Small Molecules from Human Antibody Library UsingPhage-Display Technique

(12-1) Acquisition of Antibodies that Bind to Human IL-6 in the Presenceof Small Molecules from the Library Using Bead Panning or a NegativeSelection Method

By the same method described in 2-2 and 2-3, the phage-display libraryof naïve human antibodies constructed as described in Example 2-1 wasscreened for antibodies that exhibit antigen-binding activity in thepresence of small molecules.

(12-2) Assessment of Binding Activity in the Presence of Small Moleculesby Phage ELISA

Culture supernatants containing phages were obtained from singlecolonies of E. coli obtained by the same method described in Example2-4. The phages were subjected to ELISA. By carrying out phage ELISAusing the 768 isolated clones, clones “I6NMSC1-3_(—)#03” and“I6NMSC1-3_(—)#17”, which exhibited binding activity to human IL-6 as anantigen in the presence of small molecule cocktail, were newly obtained.

(12-3) Expression and Purification of Antibodies that Bind to Human IL-6

Genes were amplified from clones I6NMSC1-3_(—)#03 and I6NMSC1-3_(—)#17that had been assessed to have antigen-binding activity in the presenceof SC by phage ELISA, using specific primers (SEQ ID NOs: 110 and 112);and their nucleotide sequences were analyzed. The heavy-chain andlight-chain sequences of I6NMSC1-3_(—)#03 are the sequences of SEQ IDNOs: 50 and 51, respectively. Meanwhile, the heavy-chain and light-chainsequences of I6NMSC1-3_(—)#17 are the sequences of SEQ ID NOs: 52 and53, respectively. The gene sequence encoding the variable region ofI6NMSC1-3_(—)#17 was inserted into an animal expression plasmid forhuman IgG1/Lambda, while the gene sequence encoding the variable regionof I6NMSC1-3_(—)#03, a known anti-human IL-6 antibody CLB8-F1 (the heavychain and light chain sequences are shown in SEQ ID NOs: 32 and 33,respectively), or an anti-human glypican-3 antibody GC413 (the heavychain and light chain sequences are shown in SEQ ID NOs: 34 and 35,respectively) as a negative control was inserted into an animalexpression plasmid for human IgG1/Kappa. The expressed antibodies werepurified by the method described in Example 3.

(12-4) Identification of Small Molecules Necessary for the Binding ofAntibody I6NMSC1-3_(—)#03 to Human IL-6

I6NMSC1-3_(—)#03 was subjected to ELISA under the nine conditionsdescribed in Table 3. Meanwhile, each small molecule was appropriatelyprepared at the concentrations shown in Table 3 using the buffersindicated in Table 4. The antigen used was biotin-labeled human IL-6.

First, a StreptaWell 96 microtiter plate (Roche) was coated at roomtemperature for one hour or more with 100 μl of PBS containing thebiotin-labeled antigen. After washing with Wash buffer to remove theunbound antigen from the plate, each well was blocked for one hour ormore with 250 μl of Blocking Buffer. After removing Blocking Buffer fromeach well, the purified IgGs were prepared to 2.5 μg/ml in Sample Buffercontaining small molecules at the final concentrations shown in Table 3,and each was aliquoted at 100 μl into the plate. The plate was allowedto stand at room temperature for one hour to allow binding of each IgGto the antigen in each well. After washing with Wash Buffer containingsmall molecules at the final concentrations shown in Table 3, anHRP-conjugated anti-human IgG antibody (BIOSOURCE) diluted with SampleBuffer containing the same small molecules was added to each well. Theplate was incubated for one hour. Following wash with Wash Buffercontaining each small molecule, the TMB single solution (ZYMED) wasadded to each well. The chromogenic reaction in the solution of eachwell was terminated by adding sulfuric acid. Then, the developed colorwas assessed by measuring absorbance at 450 nm. The composition of thebuffer used is shown in Table 4.

The measurement result is shown in FIG. 22. The result showed that theabsorbance for I6NMSC1-3_(—)#03 was markedly lower under condition 9(without small molecules) as compared to that under condition 8 (thecomplete small molecule cocktail solution). Similar to the result ofphage ELISA, this result confirmed that I6NMSC1-3_(—)#03 has theproperty that its antigen binding is altered depending on the presenceor absence of small molecules. Furthermore, the absorbance forI6NMSC1-3_(—)#03 was comparable between condition 7 (100 μM kynurenine)and condition 8; however, the absorbance was lower under otherconditions. This demonstrated that, like I6NMSC1-3_A11 described inExample 3, I6NMSC1-3_(—)#03 was an antibody that binds to human IL-6 asan antigen in the presence of kynurenine. I6NMSC1-3_(—)#03 has an aminoacid sequence different from I6NMSC1-3_A11, which demonstrates that themethod described above can be used to isolate different types ofantibodies that bind to antigens in the presence of small molecules.

(12-5) Identification of Small Molecules Necessary for the Binding ofAntibody I6NMSC1-3_(—)#17 to Human IL-6

Three types of antibodies: obtained I6NMSC1-3_(—)#17, control CLB8-F1,and negative control GC413 were subjected to ELISA under the nineconditions described in Table 3. Each small molecule was prepared to anappropriate concentration shown in Table 3 using the buffers shown inTable 4. The antigen used was biotin-labeled human IL-6.

First, a StreptaWell 96 microtiter plate (Roche) was coated at roomtemperature for one hour or more with 100 μl of PBS containing thebiotin-labeled antigen. After washing with Wash buffer to remove theunbound antigen from the plate, each well was blocked for one hour ormore with 250 μl of Blocking Buffer. After Blocking Buffer was removedfrom each well, the purified IgGs were prepared to 0.15 μg/ml in SampleBuffer containing small molecules at the final concentrations shown inTable 3, and each was aliquoted at 100 μl into the plate. The plate wasallowed to stand at room temperature for one hour to allow binding ofeach IgG to the antigen in each well. After washing with Wash Buffercontaining small molecules at the final concentrations shown in Table 3,an HRP-conjugated anti-human IgG antibody (BIOSOURCE) diluted withSample Buffer containing the same small molecules was added to eachwell. The plate was incubated for one hour. Following wash with WashBuffer containing each small molecule, the TMB single solution (ZYMED)was added to each well. The chromogenic reaction in the solution of eachwell was terminated by adding sulfuric acid. Then, the developed colorwas assessed by measuring absorbance at 450 nm. The composition of thebuffer used is shown in Table 4.

The measurement result is shown in FIG. 23. The result showed that theabsorbance for CLB8-F1 was the same regardless of the type and presenceor absence of small molecule, whereas the absorbance forI6NMSC1-3_(—)#17 was markedly lower under condition 9 (without smallmolecules) as compared to that under condition 8 (the complete smallmolecule cocktail solution). Similar to the result of phage ELISA, thisresult confirmed that I6NMSC1-3_(—)#17 has the property that its antigenbinding is altered depending on the presence or absence of smallmolecules. Furthermore, the absorbance for I6NMSC1-3_(—)#17 undercondition 1 (1 mM ATP-Na) and condition 5 (1 mM succinic acid) wascomparable to that under condition 8; however, the absorbance was lowerunder other conditions. This result suggests that I6NMSC1-3_(—)#17 is anantibody that binds to human IL-6 as an antigen in the presence ofeither ATP-Na or succinic acid. ATP is known to be released from cancercells. Succinic acid is also known to be accumulated inside and outsideof cells in a cancer cell-specific manner. The phenomenon that even inaerobic environments cancer cells metabolize in a glycolysis-dependentmanner rather than by oxidative phosphorylation is known as Warburgeffect. Both glycolysis and oxidative phosphorylation are chronicallylimited in ischemic-type cancers because of chronic insufficiency ofblood flow, and thus such cancers acquire energy in poorer conditions.Ischemic-type cancers are known to perform energy metabolism dependingon fumarate respiration, resulting in accumulation of succinic acid as afumarate metabolite (Cancer Res. (2009) 69 (11), 4918-4925).

This demonstrated that by using such method, it is possible to obtainantibodies that bind to antigens in the presence of a small moleculeother than kynurenine. Also, this showed that it is possible to obtainantibodies that bind to antigens in the presence of small molecules suchas ATP-Na and succinic acid which share a common feature that they havemultiple negative charges but are structurally different from eachother.

Example 13 Acquisition of Antibodies that Bind to Human Serum Albumin(Hereinafter Also Referred to as HSA) in the Presence of Small Moleculesby Phage-Display Techniques from Human Antibody Library

(13-1) Acquisition of Antibodies that Bind to HSA in the Presence ofSmall Molecules from Library by Bead Panning

The phage-display library of naïve human antibodies constructed asdescribed in Example 2 was screened for antibodies that exhibitHSA-binding activity in the presence of small molecules, specifically,by collecting phages displaying antibodies that in the presence of smallmolecules exhibit binding activity to HSA captured by beads. Phages werecollected from a phage suspension eluted from the beads in the absenceof small molecules. In this preparation method, biotin-labeled HSA wasused as the antigen.

Phages produced in E. coli containing the phagemid vector constructedfor phage display were purified by a conventional method. Then, a phagelibrary suspension was prepared through dialysis against TBS. Next, skimmilk was added to the phage library suspension at a final concentrationof 3%. Panning was performed using antigen-immobilized magnetic beads.The magnetic beads used were NeutrAvidin coated beads (Sera-MagSpeedBeads NeutrAvidin-coated) and Streptavidin coated beads (DynabeadsM-280 Streptavidin).

For efficient acquisition of small-molecule switch antibodies that aredependent on small molecules which can serve as a switch in cancertissues, panning was performed to enrich antibodies that bind toantigens in the presence of a mixture of such small molecules(adenosine, adenosine triphosphate (adenosine 5′-triphosphate (ATP)),inosine, kynurenine, prostaglandin E2 (PGE2), succinic acid, and lacticacid (hereinafter referred to as small molecule cocktail (SC)) but donot bind to antigens in the absence of SC.

Specifically, together with 250 pmol of the biotin-labeled antigen, SCcontaining adenosine triphosphate sodium salt (ATP-Na), adenosine,inosine, succinic acid, and lactic acid at a final concentration of 1mM, prostaglandin E2 (PGE2) at a final concentration of 1 μM, andkynurenine at a final concentration of 100 μM, which had been adjustedto be pH 7.4 with NaOH, was added and contacted with the prepared phagelibrary suspension for 60 minutes at room temperature. Then, skimmilk-blocked magnetic beads were added to the phage library suspension,and the antigen-phage complex was allowed to bind to the magnetic beadsat room temperature for 15 minutes. The beads were washed once withSC/TBS (TBS containing SC). Then, the beads were combined with 0.5 ml ofa 1 mg/ml trypsin solution. Immediately after the beads were suspendedat room temperature for 15 minutes, a phage suspension was collectedfrom the isolated beads using a magnetic stand. The collected phagesuspension was added to 10 ml of E. coli cells of strain ER2738 at thelogarithmic growth phase (OD600=0.4 to 0.7). The E. coli was incubatedat 37° C. for one hour under gentle stirring to be infected by phage.The infected E. coli was seeded in a 225 mm×225 mm plate. Then, phageswere collected from the culture medium of the seeded E. coli to preparea liquid stock of phage library.

The first round of panning was carried out to collect phages that arecapable of binding in the presence of small molecules, while the secondand subsequent rounds of panning were performed to enrich phages thatare capable of antigen binding in the presence of small molecules.Specifically, 40 pmol of the biotin-labeled antigen, SC, and NaOH wereadded to the prepared phage library suspension. Thus, the phage librarywas contacted with the antigen and small molecules for 60 minutes atroom temperature. Skim milk-blocked magnetic beads were added andallowed to bind the antigen-phage complex for 15 minutes at roomtemperature. The beads were washed with 1 ml of SC/TBST and SC/TBS.Then, the beads were combined with 0.5 ml of TBS. Immediately after thebeads were suspended at room temperature, a phage suspension wascollected from the isolated beads using a magnetic stand. Afterrepeating this treatment, the two separately eluted phage suspensionswere combined together. Then, the resultant beads were combined with 0.5ml of TBS, and stirred at room temperature for five minutes. A phagesuspension was collected from the isolated beads using a magnetic stand.The pIII protein (helper phage-derived protein pIII) that does notdisplay Fab was cleaved off from phages by adding 5 μl of 100 mg/mltrypsin to the collected phage suspension to eliminate the ability ofphages that do not display Fab to infect E. coli. The phages collectedfrom the trypsinized phage suspension were added to 10 ml of E. colicells of strain ER2738 at the logarithmic growth phase (OD600=0.4 to0.7). The E. coli was incubated with gentle stirring at 37° C. for onehour to be infected by phage. The infected E. coli was seeded in a 225mm×225 mm plate. The two types of infected E. coli obtained through tworounds of panning were mixed in equal amount at this time point. Then,phages were collected from the culture medium of the seeded E. coli toprepare a phage library suspension. Panning was performed three times toobtain antibodies that have antigen-binding activity in the presence ofsmall molecules.

(13-2) Acquisition of Antibodies that Bind to HSA in the Presence ofSmall Molecules from the Library Using a Negative Selection Method

The constructed phage-display library of naïve human antibodies wasscreened for antibodies that exhibit HSA-binding activity in thepresence of small molecules. As a first step of screening, thephage-display library of naïve human antibodies was contacted withbiotin-labeled antigen-streptavidin in the absence of small molecules toeliminate phages displaying antibodies that have HSA-binding activityeven in the absence of small molecules. Then, panning was performed inthe presence of small molecules in the same manner. Thus, screening wascarried out for antibodies that have HSA-binding activity in thepresence of small molecules. Biotin-labeled HSA was used as the antigen.

Phages were produced in E. coli containing the phagemid vectorconstructed for phage display. The produced phages were purified by aconventional method, and then a phage library suspension was prepared bydialyzing the phages against TBS. Then, skim milk was added at a finalconcentration of 3% to the phage library suspension. The magnetic beadsused were NeutrAvidin coated beads (Sera-Mag SpeedBeadsNeutrAvidin-coated) and Streptavidin coated beads (Dynabeads M-280Streptavidin). Panning was performed using biotin-labeled HSAimmobilized on magnetic beads.

Together with 250 pmol of biotin-labeled HSA, SC containing ATP-Na,adenosine, inosine, succinic acid, and lactic acid at a finalconcentration of 1 mM, PGE2 at a final concentration of 1 μM, andkynurenine at a final concentration of 100 μM, which had been adjustedto be pH 7.4 with NaOH, was added and contacted with the prepared phagelibrary solution for 60 minutes at room temperature. Then, skimmilk-blocked magnetic beads were added to the phage library solution,and allowed to bind to the complex of phage with biotin-labeled HSA for15 minutes at room temperature. The beads were washed once with SC/TBS.Then, the beads were combined with 0.5 ml of a 1 mg/ml trypsin solution.Immediately after the beads were suspended at room temperature for 15minutes, a phage suspension was collected from the isolated beads usinga magnetic stand. The collected phage suspension was added to 10 ml ofE. coli cells of strain ER2738 at the logarithmic growth phase(OD600=0.4 to 0.7). The E. coli was incubated with gentle stirring at37° C. for one hour to be infected by phage. The infected E. coli wasseeded in a 225 mm×225 mm plate. Then, phages were collected from theculture medium of the seeded E. coli to prepare a phage librarysolution.

The first round of panning was carried out to collect phages that arecapable of binding in the presence of small molecules, while the secondand subsequent rounds of panning were performed to enrich phages thatare capable of binding to biotin-labeled HSA in the presence of smallmolecules. Specifically, 250 pmol of biotin-labeled HSA was added toskim milk-blocked Sera-Mag NeutrAvidin beads and allowed to bind for 15minutes at room temperature. The beads were washed three times with TBS;and a skim milk-blocked phage library solution was added to the beads,and allowed to bind at room temperature for one hour. The beads wereisolated using a magnetic stand to collect phages that did not bind tobiotin-labeled HSA or the beads. Forty pmol of biotin-labeled HSA, SC,and NaOH were added to the collected phages. Thus, the phage library wascontacted with biotin-labeled HSA and small molecules in SC for 60minutes at room temperature. Then, skim milk-blocked magnetic beads wereadded to the mixture of biotin-labeled HSA, SC, and phage library, andthe complex of biotin-labeled HSA and phage was allowed to bind to themagnetic beads for 15 minutes at room temperature. The beads were washedwith 1 ml of SC/TBST and SC/TBS. Then, 0.5 ml of a 1 mg/ml trypsinsolution was added to the mixture. After the mixed solution was stirredfor 20 minutes at room temperature, phages were collected from the beadsseparated using a magnetic stand. The collected phages were added to 10ml of E. coli cells of strain ER2738 at the logarithmic growth phase(OD600=0.4 to 0.7). The E. coli was incubated with gentle stirring at37° C. for one hour to be infected by phage. The infected E. coli wasseeded in a 225 mm×225 mm plate. Panning was performed three times toobtain antibodies that have binding activity to biotin-labeled HSA inthe presence of small molecules.

(13-3) Assessment of Binding Activity in the Presence of Small Moleculesby Phage ELISA

Culture supernatants containing phages were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained by the method described above. Thecollected culture supernatants were treated by ultrafiltration usingNucleoFast 96 (MACHEREY-NAGEL). 100 μl of the culture supernatants wereadded to each well, and the NucleoFast 96 was centrifuged (4500 g for 45minutes) to remove flow-through. One hundred μl of H₂O was added to eachwell, and again the NucleoFast 96 was centrifuged (4500 g for 30minutes) for washing. After 100 μl of TBS was added, the NucleoFast 96was allowed to stand for five minutes at room temperature. Finally,phage solution contained in the supernatant of each well was collected.

The purified phages, to which TBS or SC/TBS was added, were subjected toELISA by the following procedure. A StreptaWell 96 microtiter plate(Roche) was coated overnight with 100 μl of TBS containingbiotin-labeled HSA. After biotin-labeled HSA was removed by washing eachwell of the plate with TBST, the wells were blocked with 250 μl of 2%skim milk/TBS for one hour or more. 2% skim milk/TBS was removed, andthen the prepared, purified phages were added to each well. The platewas allowed to stand at room temperature for one hour to allow bindingof antibody-displaying phages to biotin-labeled HSA in each well in thepresence or absence of SC. After washing with TBST or SC/TBST, anHRP-conjugated anti-M13 antibody (Amersham Pharmacia Biotech) dilutedwith TBS or SC/TBS was added to each well. The plate was incubated forone hour. Following wash with TBST or SC/TBST, the TMB single solution(ZYMED) was added to each well. The chromogenic reaction in the solutionof each well was terminated by adding sulfuric acid. Then, the developedcolor was assessed by measuring absorbance at 450 nm.

Clone HSNMSC1-4_(—)#22, which has binding activity to HSA as antigen inthe presence of the small molecule cocktail, was obtained by carryingout phage ELISA using the 782 isolated clones.

(13-4) Expression and Purification of HSA-Binding Antibodies

Genes were amplified from clone HSNMSC1-4_(—)#22 which had been assessedto have binding activity to biotin-labeled HSA in the presence of SC bythe phage ELISA described in (13-3), using specific primers (SEQ ID NOs:110 and 112). The nucleotide sequences of the genes were analyzed (theheavy chain and light chain sequences are represented by SEQ ID NOs: 54and 55, respectively). Genes encoding the variable regions ofHSNMSC1-4_(—)#22 were inserted into an animal expression plasmid forhuman IgG1/Lambda. Meanwhile, genes encoding the variable regions of thenegative control anti-human glypican-3 antibody GC413 (the heavy chainand light chain are represented by SEQ ID NOs: 34 and 35, respectively)were inserted into an animal expression plasmid for human IgG1/Kappa.The expressed antibodies were purified by the method described inExample 3.

(13-5) Identification of Small Molecules Necessary for Binding of theObtained Antibodies to HSA

Two types of antibodies obtained: HSNMSC1-4_(—)#22 and GC413, weresubjected to ELISA under the nine conditions described in Table 3.Meanwhile, each small molecule was appropriately prepared at theconcentrations shown in Table 3 using the buffers indicated in Table 19.Biotin-labeled HSA was used as the antigen.

TABLE 19 Wash buffer 10 mM ACES, 150 mM NaCl, 0.05% Tween20, pH7.4Blocking Buffer 10 mM ACES, 150 mM NaCl, 2% Skim Milk, pH7.4 SampleBuffer 10 mM ACES, 150 mM NaCl, Small molecule, pH7.4

First, a StreptaWell 96 microtiter plate (Roche) was coated at roomtemperature for one hour or more with 100 μl of PBS containingbiotin-labeled HSA. After washing with Wash buffer to remove unboundbiotin-labeled HSA from the plate, each well was blocked for one hour ormore with 250 μl of Blocking Buffer. After Blocking Buffer was removedfrom each well, the purified IgGs were prepared to 2.5 μg/ml in SampleBuffer containing small molecules at the final concentrations shown inTable 3, and each was aliquoted at 100 μl into the plate. The plate wasallowed to stand at room temperature for one hour to allow binding ofeach IgG to biotin-labeled HSA in each well. After washing with WashBuffer containing small molecules at the final concentrations shown inTable 3, an HRP-conjugated anti-human IgG antibody (BIOSOURCE) dilutedwith Sample Buffer containing the same small molecules was added to eachwell. The plate was incubated for one hour. Following wash with WashBuffer containing each small molecule, the TMB single solution (ZYMED)was added to each well. The chromogenic reaction in the solution of eachwell was terminated by adding sulfuric acid. Then, the developed colorwas assessed by measuring absorbance at 450 nm. The composition of thebuffer used is shown in Table 19.

The measurement result is shown in FIG. 24. The result showed that theabsorbance for HSNMSC1-4_(—)#22 was markedly lower under condition 9(without small molecules) as compared to that under condition 8 (thecomplete small molecule cocktail solution). Similar to phage ELISA, thisresult confirmed that HSNMSC1-4_(—)#22 has the property that its antigenbinding is altered depending on the presence or absence of smallmolecules. Meanwhile, the absorbance for HSNMSC1-4_(—)#22 was the samebetween condition 2 (1 mM adenosine) and condition 8, but it wasmarkedly lower under other conditions. This result demonstrated thatHSNMSC1-4_(—)#22 is an antibody that binds to HSA as an antigen in thepresence of adenosine. Thus, it was demonstrated that such method can beused to isolate antibodies that bind to antigens in the presence ofsmall molecules other than kynurenine.

Example 14 Acquisition of Antibodies that Bind to Human IL-6 Receptor(hIL-6R) in the Presence of Small Molecules from Human Antibody LibraryUsing Phage Display Techniques

(14-1) Acquisition of Antibodies that Bind to hIL-6R in the Presence ofSmall Molecules from the Library of Naïve Human Antibodies by BeadPanning

The phage-display library of naïve human antibodies constructed asdescribed in Example 2 was screened for antibodies that exhibithIL-6R-binding activity in the presence of small molecules,specifically, by collecting phages displaying antibodies that in thepresence of small molecules exhibit binding activity to hIL-6R capturedby beads. Phages were collected from a phage eluate eluted from thebeads in the absence of small molecules. In this preparation method,biotin-labeled hIL-6R was used as the antigen.

Phages produced in E. coli containing the phagemid vector constructedfor phage display were purified by a conventional method. Then, a phagelibrary solution was prepared by dialyzing the phages against TBS. Next,BSA was added at a final concentration of 4% to the phage librarysolution. Panning was performed using antigen-immobilized magneticbeads. The magnetic beads used were NeutrAvidin coated beads (Sera-MagSpeedBeads NeutrAvidin-coated) and Streptavidin coated beads (DynabeadsM-280 Streptavidin).

For efficient acquisition of small molecule switch antibodies that aredependent on small molecules which can serve as a switch in cancertissues, panning described in (2-2) was performed to enrich antibodiesthat bind to antigens in the presence of SC but do not bind to antigensin the absence of SC.

Specifically, together with 250 pmol of biotin-labeled antigen, SCprepared as described in (2-2) was added and contacted with the preparedphage library solution at room temperature for 60 minutes. Then, thephage library solution was added to BSA-blocked magnetic beads, and theantigen-phage complex was allowed to bind to the magnetic beads for 15minutes at room temperature. The beads were washed once with SC/TBS (TBScontaining SC). Then, the beads were combined with 0.5 ml of a 1 mg/mltrypsin solution. Immediately after the beads were suspended at roomtemperature for 15 minutes, the phage solution was collected from theisolated beads using a magnetic stand. The collected phage solution wasadded to 10 ml of E. coli cells of strain ER2738 at the logarithmicgrowth phase (OD600=0.4 to 0.7). The E. coli was incubated with gentlestirring at 37° C. for one hour to be infected by phage. The infected E.coli was seeded in a 225 mm×225 mm plate. Then, phages were collectedfrom the culture medium of the seeded E. coli to prepare a liquid stockof phage library.

Panning was performed as described in (2-2), except adding 10 μl of 100mg/ml trypsin to cleave the pIII protein (helper phage-derived pIIIprotein) from phages that do not display Fab in order to eliminate theability of the phages that do not display Fab to infect E. coli.

(14-2) Acquisition of Antibodies that Bind to hIL-6R in the Presence ofSmall Molecules from the Naïve Human Antibody Library Using a NegativeSelection Method

The constructed phage-display library of naïve human antibodies wasscreened for antibodies that exhibit hIL-6R-binding activity in thepresence of small molecules. As a first step of screening, thephage-display library of naïve human antibodies was contacted withbiotin-labeled antigen-streptavidin in the absence of small molecules toeliminate phages displaying antibodies that have hIL-6R-binding activityeven in the absence of small molecules. Then, panning was performed inthe presence of small molecules in the same manner to screen forantibodies that have hIL-6R-binding activity in the presence of smallmolecules. The antigen used was biotin-labeled hIL-6R. Then, a phagelibrary solution was prepared by the method described in (2-3) usingbiotin-labeled hIL-6R as an antigen.

(14-3) Assessment of Binding Activity in the Presence of Small Moleculesby Phage ELISA

Culture supernatants containing phages were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained as described in (14-2). Phages purified bythe method described in (2-4) were subjected to ELISA by the followingprocedure. A StreptaWell 96 microtiter plate (Roche) was coatedovernight with 100 μl of TBS containing biotin-labeled hIL-6R. Afterbiotin-labeled hIL-6R was removed by washing each well of the plate withTBST, the wells were blocked with 250 μl of 2% skim milk/TBS for onehour or more. 2% skim milk/TBS was removed, and then the prepared,purified phages were added to each well. The plate was allowed to standat room temperature for one hour to allow binding of antibody-displayingphages to biotin-labeled hIL-6R in each well in the presence or absenceof SC. After washing with TBST or SC/TBST, an HRP-conjugated anti-M13antibody (Amersham Pharmacia Biotech) diluted with TBS or SC/TBS wasadded to each well. The plate was incubated for one hour. Following washwith TBST or SC/TBST, the TMB single solution (ZYMED) was added to eachwell. The chromogenic reaction in the solution of each well wasterminated by adding sulfuric acid. Then, the developed color wasassessed by measuring absorbance at 450 nm.

Clones 6RNMSC1-2_F02 and 6RNMSC1-3_G02, which have binding activity tohIL-6R as an antigen in the presence of a small molecule cocktail, wereobtained by carrying out phage ELISA using 960 isolated clones.

(14-4) Expression and Purification of Antibodies that Bind to hIL-6R

Genes were amplified using specific primers (SEQ ID NOs: 110 and 112)from clones 6RNMSC1-2_F02 and 6RNMSC1-3_G02, which had been assessed tohave binding activity to biotin-labeled hIL-6R in the presence of SC bythe phage ELISA described in (14-3). The nucleotide sequences of thegenes were analyzed (6RNMSC1-2_F02: the heavy chain and light chainsequences are shown in SEQ ID NOs: 86 and 87, respectively;6RNMSC1-3_G02: the heavy chain and light chain sequences are shown inSEQ ID NOs: 88 and 89, respectively). Genes encoding the variableregions of 6RNMSC1-2_F02 and 6RNMSC1-3_G02, and those of the negativecontrol anti-human glypican-3 antibody GC413 (the heavy chain and lightchain are SEQ ID NOs: 34 and 35, respectively) were inserted into ananimal expression plasmid for human IgG1/Kappa. The expressed antibodieswere purified by the method described in Example 3.

(14-5) Identification of Small Molecules Necessary for Binding of theObtained Antibodies to Hil-6R

Three types of antibodies obtained: 6RNMSC1-2_F02 and 6RNMSC1-3_G02, andGC413 were subjected to ELISA under the nine conditions described inTable 3. Meanwhile, each small molecule was appropriately prepared atthe concentrations shown in Table 3 using the buffers indicated in Table19. The antigen used was biotin-labeled hIL-6R.

First, a StreptaWell 96 microtiter plate (Roche) was coated at roomtemperature for one hour or more with 100 μl of PBS containingbiotin-labeled hIL-6R. After washing with Wash buffer to remove unboundbiotin-labeled hIL-6R from the plate, each well was blocked for one houror more with 250 μl of Blocking Buffer. After Blocking Buffer wasremoved from each well, the purified IgGs were prepared to 2.5 μg/ml inSample Buffer containing small molecules at the final concentrationsshown in Table 3, and each was aliquoted at 100 μl into the plate. Theplate was allowed to stand at room temperature for one hour to allowbinding of each IgG to biotin-labeled hIL-6R in each well. After washingwith Wash Buffer containing small molecules at the final concentrationsshown in Table 3, an HRP-conjugated anti-human IgG antibody (BIOSOURCE)diluted with Sample Buffer containing the same small molecules was addedto each well. The plate was incubated for one hour. Following wash withWash Buffer containing each small molecule, the TMB single solution(ZYMED) was added to each well. The chromogenic reaction in the solutionof each well was terminated by adding sulfuric acid. Then, the developedcolor was assessed by measuring absorbance at 450 nm. The composition ofthe buffer used is shown in Table 19.

The measurement result is shown in FIGS. 28 and 29. When 6RNMSC1-2_F02or 6RNMSC1-3G02 was used, the absorbance was markedly lower undercondition 9 (in the absence of small molecules) as compared to undercondition 8 (the complete small molecule cocktail solution). This resultconfirmed that 6RNMSC1-2_F02 and 6RNMSC1-3 G02 have the property thattheir antigen binding is altered depending on the presence or absence ofsmall molecules. Meanwhile, when 6RNMSC1-2_F02 was used, the absorbancewas the same between condition 7 (100 μM kynurenine) and condition 8,but the absorbance was markedly lower under other conditions, whichshows that 6RNMSC1-2_F02 is an antibody that binds to hIL-6R as anantigen in the presence of kynurenine (FIG. 28). Furthermore, when6RNMSC1-3G02 was used, the absorbance was the same between condition 1(1 mM ATP-Na) and condition 8, but the absorbance was markedly lowerunder other conditions, showing that 6RNMSC1-3G02 is an antibody thatbinds to hIL-6R as an antigen in the presence of ATP (FIG. 29). It wasthus demonstrated that the method described above can be used to isolateat one time multiple antibodies whose antigen-binding activity isaltered in the presence of a different small molecule.

Example 15 Characterization of Antibody 6RNMSC1-2_F02

(15-1) ELISA Assessment of hIL6R-Binding Activity in the Presence ofAmino Acids and Amino Acid Metabolites Other than Kynurenine

Antibody 6RNMSC1-2_F02 obtained as described in Example 14, which bindsto hIL-6R in the presence of small molecules, is an antibody that bindsto hIL-6R in the presence of kynurenine. Amino acid metabolites such astryptophan metabolites described in Example 11 were assessed as towhether they are preferable as a non-limiting embodiment of cancertissue-specific compounds, particularly cancer cell-specificmetabolites, for use in the present invention.

6RNMSC1-2_F02 described in Example 14, which has antigen-bindingactivity in the presence of kynurenine, and negative control GC413 weresubjected to ELISA under the seven conditions described in Table 18.Meanwhile, each small molecule was appropriately prepared at theconcentrations shown in Table 18 using the buffers indicated in Table 4.The antigen used was biotin-labeled hIL-6R. ELISA was carried out usingthe method described in Example 11.

The measurement result is shown in FIG. 30. When 6RNMSC1-2_F02 was used,the absorbance was markedly lower under condition 7 (in the absence ofsmall molecules) as compared to under condition 1 (kynurenine solution).Similarly, 6RNMSC1-2_F02 showed the same high absorbance under condition5 (3-hydroxykynurenine solution) as under condition 1, suggesting thatit is an antibody that binds to hIL-6R as an antigen not only in thepresence of kynurenine but also in the presence of a kynureninemetabolite. Furthermore, the absorbance for 6RNMSC1-2_F02 was markedlylower under other conditions, suggesting that 6RNMSC1-2_F02 is anantibody that does not bind to hIL-6R as an antigen in the presence oftryptophan, a kynurenine precursor. The expression level of IDO, anenzyme that metabolizes tryptophan to produce kynurenine, is elevated incancer microenvironment. Thus, antibodies that bind to antigens in thepresence of kynurenine or its metabolite but not in the presence oftryptophan are expected to be important as antibodies that bind toantigens only in cancer microenvironment. This suggests that the samemethod can be used to obtain antibodies that bind to an antigen ofinterest not only in the presence of a single amino acid metabolite butalso in the presence of multiple, structurally different amino acidmetabolites.

(15-2) Assessment of Kynurenine for its Effect on Human IL6 ReceptorBinding by Surface Plasmon Resonance

Biacore T200 (GE Healthcare) was used to analyze the interaction of6RNMSC1-2_F02 with human IL6 receptor (IL-6R) in antigen-antibodyreaction. Sensor chip CM5 (GE Healthcare) was immobilized with anappropriate amount of protein A (Invitrogen) by amine coupling. Theantibody of interest was captured by the chip to allow interaction withIL-6R as an antigen. The running buffer used was 20 mmol/l ACES, 150mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4. The interaction with theantigen IL-6R was assayed at 25° C. The buffers used to dilute IL-6Rwere the running buffer itself, and a buffer prepared by adding 100μmol/l kynurenine to the running buffer, and in addition a bufferprepared as a control by adding 10 mmol/l ATP to the running buffer.

A diluted IL-6R solution and a running buffer as a blank were injectedat a flow rate of 10 μl/min for one minute to allow interaction of IL-6Rwith 6RNMSC1-2_F02 captured on the sensor chip. Then, the running bufferwas injected at a flow rate of 10 μl/min for one minute. Afterobservation of IL-6R dissociation from the antibody, 10 mmol/lglycine-HCl (pH 1.5) was injected at a flow rate of 30 μl/min for 30seconds to regenerate the sensor chip. The dissociation constant K_(D)(M) of 6RNMSC1-2_F02 was calculated for IL-6R based on the associationrate constant ka (1/Ms) and dissociation rate constant kd (1/s), both ofwhich are kinetic parameters calculated from the sensorgram obtained bythe measurement. Each parameter was calculated using the Biacore T200Evaluation Software (GE Healthcare).

Sensorgrams obtained by this measurement for the interaction between6RNMSC1-2_F02 and 1 μmol/l IL-6R in the presence of 100 μmol/lkynurenine and in the presence or absence of 10 mmol/l ATP are shown inFIG. 31. As shown in FIG. 31, 6RNMSC1-2_F02 binds to IL-6R in thepresence of 100 μmol/l kynurenine; however, its IL-6R binding was notdetectable in the absence of kynurenine. This demonstrates that6RNMSC1-2_F02 has the property that it binds to IL-6R via kynurenine asa switch. Meanwhile, the dissociation constant K_(D) of 6RNMSC1-2_F02was 1.5 μmol/l in the presence of 100 μmol/l kynurenine.

(15-3) Effect of Kynurenine as a Switch on Dissociation of Antibodiesfrom IL-6R

Biacore T200 (GE Healthcare) was used to evaluate whether 6RNMSC1-2_F02which binds to IL-6R in the presence of kynurenine dissociates in akynurenine concentration-dependent manner in the presence of kynurenine.The running buffers used were 20 mmol/l ACES, 150 mmol/lNaCl, 0.05%(w/v) Tween20, pH 7.4, and 20 mmol/l ACES, 150 mmol/l NaCl, 0.05% (w/v)Tween20, pH 7.4, 100 μmol/l kynurenine, and assay was carried out at 25°C. IL-6R was immobilized onto Sensor chip CM5 by amine coupling; and6RNMSC1-2_F02 was diluted to 5 μg/ml with 20 mmol/lACES, 150 mmol/lNaCl, 0.05% (w/v) Tween20, pH 7.4, containing 100 μmol/l kynurenine, andit was interacted as an analyte for 180 seconds. Then, the dissociationof IL-6R was monitored under each running buffer condition. In order tocompare the degree of dissociation between the respective running bufferconditions, the values were normalized by taking as 100 the amount of6RNMSC1-2_F02 bound to IL-6R in the presence of 100 μmol/l kynurenineand compared. A sensorgram that represents the interaction between6RNMSC1-2_F02 and IL-6R after normalization is shown in FIG. 32. Theresult shown in FIG. 32 demonstrates that 6RNMSC1-2_F02 has the propertythat it binds to IL-6R in the presence of kynurenine and then rapidlydissociates from IL-6R in the absence of kynurenine. Specifically, thekynurenine-mediated regulation on binding of the antibody to IL-6R wasdemonstrated to be reversible.

(15-4) Assessment of Kynurenine Concentration Effect on IL-6R Binding

Next, Biacore T200 (GE Healthcare) was used to assess the effect ofkynurenine concentration on the antigen-antibody reaction between6RNMSC1-2_F02 and IL-6R. The running buffer used was 20 mmol/l ACES, 150mmol/l NaCl, 0.05% (w/v) Tween20, pH 7.4. The antigen-antibody reactionbetween 6RNMSC1-2_F02 and human IL-6R was assayed at 25° C. IL-6R wasimmobilized onto sensor chip CM5 by amine coupling; and 6RNMSC1-2_F02was diluted to 1 μg/ml with 20 mmol/lACES, 150 mmol/l NaCl, 0.05% (w/v)Tween20, pH 7.4 containing various concentrations of kynurenine, and itwas allowed to interact as an analyte for 180 seconds to observe changesin the amount of binding. The result is shown in FIG. 33. This resultdemonstrated that the higher the concentration of kynurenine serving asa switch, the greater the amount of 6RNMSC1-2_F02 bound to IL-6R.

Meanwhile, since IL-6R is immobilized on a sensor chip in this assaysystem, 6RNMSC1-2_F02 is thought to bind in a divalent manner. In suchan assay system where 6RNMSC1-2_F02 recognizes IL-6R in a divalentmanner, the amount of 6RNMSC1-2_F02 bound to IL-6R was also observed toincrease with a higher kynurenine concentration. This resultdemonstrated that 6RNMSC1-2_F02 has the property that it binds to IL-6Rvia kynurenine as a switch also in divalent binding.

These results demonstrated that 6RNMSC1-2_F02 is an antibody that bindsto IL-6R in the presence of kynurenine with kynurenine as a switch, butis dissociated from IL-6R in the absence of kynurenine. Furthermore, itwas confirmed that it is possible to have full ON/OFF control of6RNMSC1-2_F02 so that it does not demonstrate IL-6R-binding activity inthe absence of kynurenine. The switch function was expected to beachieved in the manner such as shown in FIG. 2.

(15-5) Effect of Kynurenine on the ADCC Activity of 6RNMSC1-2_F02

The genes encoding the variable regions of 6RNMSC1-2_F02 determined asdescribed in Example 14 were inserted into an animal expression plasmidfor human IgG1/Kappa, which comprises an antibody heavy-chain constantregion comprising the sequence of SEQ ID NO: 90 and a light-chain kappaconstant region sequence comprising the sequence of SEQ ID NO: 91. Therespective genes encoding the variable regions of a known anti-humanIL-6R antibody, MRA (the heavy chain and light chain sequences are shownin SEQ ID NOs: 92 and 93, respectively), were also inserted into theanimal expression plasmid for human IgG1/Kappa, which has theabove-described constant regions (SEQ ID NOs: 90 and 91). Antibodieswere expressed using the method described below. FreeStyle 293-F(Invitrogen) which was derived from human fetal kidney cells weresuspended at a cell density of 1.33×10⁶ cells/ml in the FreeStyle 293Expression Medium (Invitrogen), and aliquoted at 3 ml into each well ofa 6-well plate. The plasmid DNA was transfected into the cells bylipofection. From the culture supernatants after four days of culture ina CO₂ incubator (37° C., 8% CO₂, 90 rpm), antibodies were purified by amethod known to those skilled in the art using rProtein A Sepharose™Fast Flow (Amersham Biosciences). Absorbance of the purified antibodysolutions was measured at 280 nm using a spectrophotometer. From valuesobtained by the measurement, the concentrations of purified antibodieswere calculated using an extinction coefficient determined by the PACEmethod (Protein Science (1995) 4, 2411-2423).

6RNMSC1-2_F02 antibody which has an antigen-binding activity in thepresence of kynurenine as described in Example 14 was assessed for itsbinding to soluble hIL-6R. As a first step in assessing the ADCCactivity of 6RNMSC1-2_F02 to hIL-6R-expressing cells, 6RNMSC1-2_F02 wasevaluated as to whether it has the ability to bind to membrane-typehIL-6R expressed in hIL-6R-expressing cells. Specifically, the bindingof 6RNMSC1-2_F02 to cells of the BaF/hIL-6R line (WO2012/073992) wasassayed and analyzed using a flow cytometer. An appropriate number ofBaF/hIL-6R cells were prepared and blocked with PBS containing 2% FBS onice for one hour or more. Supernatant was removed from the blocked cellsby centrifugation, and 100 μl of 6RNMSC1-2_F02 or the control antibodyMRA (the heavy chain and light chain sequences are shown in SEQ ID NOs:92 and 93, respectively) was added under two conditions: in the presenceor absence of a final concentration of kynurenine at 100 μM. In thisstep, the antibodies were contacted with cell-membrane hIL-6R on ice for30 minutes. The antibody-cell complex was washed with Wash Buffercontaining kynurenine or with Wash Buffer that does not containkynurenine. Then, in the presence or absence of kynurenine, the complexwas contacted with a secondary antibody (Beckman Coulter IM1627) thatrecognizes the antibody constant region. After 30 minutes of incubationwith the antibody on ice, the cells were again washed with Wash Bufferand then re-suspended in PBS/2% FBS. The binding of 6RNMSC1-2_F02 to theprepared cells was assayed and analyzed using the BD FACS cant II FlowCytometer (BD).

The assay result was shown in FIG. 34. With the control antibody MRA,fluorescence was detectable regardless of the presence or absence ofkynurenine. In contrast, for 6RNMSC1-2_F02, fluorescence shift wasobserved for the first time in the presence of 100 μM kynurenine;however, fluoresce was not detectable in the absence of kynurenine. Thisdemonstrates that 6RNMSC1-2_F02 is an antibody that has the ability tobind to hIL-6R expressed on cell membrane in the presence of kynurenine.

In general, natural antibodies bind at their Fab directly to antigens ontarget cells and their Fc binds to FcγR on effector cells, resulting ininduction of cytotoxic activity (ADCC activity) of effector cellsagainst the target cells. In this context, whether ADCC activity isexerted against hIL-6R-expressing cells upon binding of 6RNMSC1-2_F02 tohIL-6R in the presence of kynurenine was assessed by the methoddescribed below.

Variant 6RNMSC1-2_F02 with increased effector activity (the heavy chainand light chain sequences are shown in SEQ ID NOs: 94 and 91,respectively) was used. At various concentrations of 6RNMSC1-2_F02, ADCCactivity against hIL-6R-expressing cells was assayed in the presence orabsence of kynurenine according to the method described in ReferenceExample 1. The assay result is shown in FIG. 35.

The assay result confirmed that in the presence of kynurenine, ADCCactivity against hIL-6R-expressing cells was induced by 6RNMSC1-2_F02 inan antibody concentration-dependent manner. The finding demonstratesthat the antigen binding of the antibody via kynurenine induces ADCCactivity against antigen-expressing cells and, in terms of the functionvia antitumor activity such as ADCC activity, antibodies that bind toantigens in the presence of small molecules as a switch can also beregulated by the presence of small molecules serving as a switch.

Furthermore, there is a difference in the kynurenine concentrationbetween normal and tumor tissues, and thus it is preferable that theADCC activity of an antibody is exerted against antigen-expressing tumorcells only at the kynurenine concentration in tumor tissues, while atthe normal-tissue kynurenine concentration, the ADCC activity isimpaired or not exerted. Then, 6RNMSC1-2_F02 was assessed for ADCCactivity against hIL-6R-expressing cells at various kynurenineconcentrations according to the method described in Reference Example 2.The assay result is shown in FIG. 36. The assay result demonstrated thatADCC activity against hIL-6R-expressing cells was included with6RNMSC1-2_F02 in a kynurenine concentration-dependent manner.Furthermore, in contrast to the approximately 10% ADCC activity at 4 to6 μM which is considered to be the kynurenine concentration in normaltissues, the ADCC activity at 30 to 40 μM which is considered to be thekynurenine concentration in tumor tissues was about 25%.

Based on these results, with 6RNMSC1-2_F02, the ADCC activity againsthIL-6R-expressing cells was weak in normal tissues where the kynurenineconcentration is low; and in tumor tissues where the concentration ishigh, the ADCC activity of 6RNMSC1-2_F02 against hIL-6R-expressing cellswas greater. This suggests that by administering an antibody that useskynurenine as a switch, the cytotoxicity to normal tissues expressing atarget antigen can be reduced without impairing the pharmaceuticaleffect against tumor tissues expressing the target antigen.

(15-6) Assessment of the Obtained Antibody for its Binding Activity tohIL-6R in Mouse Serum by Igg ELISA

Antibody 6RNMSC1-2_F02 obtained as described in Example 14, which bindsto hIL-6R in the presence of small molecules, is an antibody that bindsto hIL-6R in the presence of kynurenine. So far, 6RNMSC1-2_F02 has beenassessed for its antigen-binding ability in buffers such as PBS and TBS.Many unknown small molecules including amino acids are considered toexist in mouse serum, and one cannot rule out the possibility that suchsmall molecules affect the antigen binding of 6RNMSC1-2_F02. Thus,6RNMSC1-2_F02 was assessed for its antigen-binding ability in mouseserum.

As described in Example 14, antibody 6RNMSC1-2_F02 which hasantigen-binding activity in the presence of kynurenine, and the knownanti-hIL-6R antibody MRA were subjected to ELISA under the twoconditions described in Table 20. The antigen used was biotin-labeledhIL-6R.

TABLE 20 Condition Buffer composition 1 Mouse serum 2 Mouse serum, 100μM Kynurenine

First, a StreptaWell 96 microtiter plate (Roche) was coated at roomtemperature for one hour or more with 100 μl of PBS containing thebiotin-labeled antigen. After washing with Wash buffer to remove unboundantigen from the plate, each well was blocked for one hour or more with250 μl of Blocking Buffer. After Blocking Buffer was removed from eachwell, each of the purified IgGs was prepared to 2.5 μg/ml undercondition 2 shown in Table 20, and aliquoted at 100 μl into the plate.The plate was allowed to stand at room temperature for one hour to allowbinding of each IgG to the antigen in each well. After washing with WashBuffer containing 100 μM kynurenine, an HRP-conjugated anti-human IgGantibody (BIOSOURCE) diluted with Sample Buffer containing kynureninewas added to each well. The plate was incubated for one hour. Followingwash with Wash Buffer containing kynurenine, the TMB single solution(ZYMED) was added to each well. The chromogenic reaction in the solutionof each well was terminated by adding sulfuric acid. Then, the developedcolor was assessed by measuring absorbance at 450 nm.

The measurement result is shown in FIG. 37. With MRA, the absorbance wasthe same regardless of the presence or absence of kynurenine. Incontrast, when 6RNMSC1-2_F02 was used, the absorbance was markedly lowerunder condition 1 (mouse serum without kynurenine) as compared to undercondition 2 (mouse serum in the presence of kynurenine) shown in Table20. This suggests that 6RNMSC1-2_F02 is an antibody that binds to hIL-6Ras an antigen in the presence of kynurenine without being affected byunknown small molecules in mouse serum.

Example 16 Acquisition of Antibodies that Bind to Antigens in theAbsence of Adenosine or ATP from Antibody Library Using Phage-DisplayTechniques

(16-1) Acquisition of Antibodies Whose Antigen Binding is Inhibited inthe Presence of Small Molecules from a Library Using a Mixture ofAdenosine and ATP

Antibodies that bind to target antigens in the presence of smallmolecules serving as a switch were obtained as described in Examplesabove. In the experiment described in this Example, the presentinventors attempted to obtain antibodies that bind to target antigens inthe absence of small molecules.

Antibodies that exhibit antigen-binding activity in the absence ofadenosine and/or ATP but whose binding ability is impaired in thepresence of adenosine and/or ATP were obtained from a constructedphage-display library of rationally designed antibodies. As a first stepto isolate antibodies, a phage-display library of antibodies wascontacted with biotinylated adenosine and ATP-NeutrAvidin to collect aphage-display library of antibodies that bind to adenosine and/or ATP.Then, the phage-display antibody library was contacted with biotinylatedantigen-streptavidin in the absence of adenosine and ATP to collectantibodies that bind to antigens in the absence of adenosine and ATP.Panning was performed in the alternating manner described above toscreen for antibodies that have binding activity to both antigen andadenosine and/or ATP. In the presence of adenosine and ATP, the antigenbinding of antibodies with such properties was expected to be inhibitedby binding of adenosine and/or ATP to the antibodies.

Phages were produced in E. coli containing the phagemid vectorconstructed for phage display. To the culture medium of E. coli in whichphage production was carried out, 2.5 M NaCl/10% PEG was added toprecipitate phages. The precipitated phage fraction was diluted with TBSto prepare a phage library solution. Then, BSA was added at a finalconcentration of 4% to the phage library solution. Panning was performedusing antigen-immobilized magnetic beads. The magnetic beads used wereNeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) andStreptavidin-coated beads (Dynabeads M-280 Streptavidin).

500 pmol of biotinylated ATP, 2′-adenosine-PEG-Biotin, and5′-adenosine-PEG-Biotin were added to the prepared phage librarysolution. Thus, the phage library solution was contacted with adenosineand ATP for 60 minutes at room temperature. Then, BSA-blocked magneticbeads were added to the phage library solution and the complex of phagewith adenosine and/or ATP was allowed to bind to the magnetic beads atroom temperature for 15 minutes. The beads were washed once with TBS,and then 0.5 ml of a 1 mg/ml trypsin solution was added to the beads.

Immediately after the beads were suspended at room temperature for 15minutes, a phage solution was collected from the isolated beads using amagnetic stand. The collected phage solution was added to 10 ml of E.coli cells of strain ER2738 at the logarithmic growth phase (OD600=0.4to 0.7). The E. coli was incubated with gentle stirring at 37° C. forone hour to be infected by phage. The infected E. coli was seeded in a225 mm×225 mm plate. Then, phages were collected from the culture mediumof the seeded E. coli to prepare a phage library solution.

The second round of panning was performed to enrich phages capable ofbinding to the biotinylated antigen in the absence of adenosine and ATP.Specifically, 250 pmol of biotinylated antigen was added to the preparedphage library solution. Thus, the phage library solution was contactedwith the antigen for 60 minutes at room temperature. Then, BSA-blockedmagnetic beads were added to the phage library solution, and theantigen-phage complex was allowed to bind to the magnetic beads at roomtemperature for 15 minutes. The beads were washed twice with TBST andonce with TBS. Then, 0.5 ml of a 1 mg/ml trypsin solution was added tothe beads. Immediately after the beads were suspended at roomtemperature for 15 minutes, a phage solution was collected from theisolated beads using a magnetic stand. The collected phage solution wasadded to 10 ml of E. coli cells of strain ER2738 at the logarithmicgrowth phase (OD600=0.4 to 0.7). The E. coli was incubated with gentlestirring at 37° C. for one hour to be infected by phage. The infected E.coli was seeded in a 225 mm×225 mm plate. Then, phages were collectedfrom the culture medium of the seeded E. coli to prepare a phage librarysolution.

At subsequent odd-numbered rounds, panning was performed in the samemanner as the first-round panning. However, the number of bead washeswith TBST and TBS was increased to three times and twice, respectively.

At subsequent even-numbered rounds, panning was performed in the samemanner as the second-round panning. However, in the fourth andsubsequent rounds of panning, the biotinylated antigen was reduced to 40pmol, and the number of bead washes with TBST and TBS was increased tothree times and twice, respectively.

(16-2) Assessment of Binding Activity in the Presence of Small Moleculesby Phage ELISA

Culture supernatants containing phages were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained by the method described above. Thecollected culture supernatants were ultrafiltrated using NucleoFast 96(MACHEREY-NAGEL). 100 μl of the culture supernatants were added to eachwell, and the NucleoFast 96 was centrifuged (4500 g for 45 minutes) toremove flow-through. 100 μl of H₂O was added to each well, and again theNucleoFast 96 was washed by centrifugation (4500 g for 30 minutes).After 100 μl of TBS was added, the NucleoFast 96 was allowed to stand atroom temperature for five minutes. Finally, a phage solution wascollected from the supernatant in each well.

Purified phages, to which TBS, or ATP and adenosine/TBS had been added,were subjected to ELISA by the following procedure. A StreptaWell 96microtiter plate (Roche) was coated overnight with 100 μl of TBScontaining a biotin-labeled antigen. After the antigen was removed fromeach well of the plate by washing with TBST, the wells were blocked with250 μl of 2% skim milk/TBS for one hour or more. 2% skim milk/TBS wasremoved, and then the prepared, purified phages were added to each well.The plate was allowed to stand at 37° C. for one hour to allow bindingof antibody-displaying phages to antigens in each well in the presenceor absence of 10 mM adenosine and ATP. After washing with TBST or 10 mM(ATP and adenosine)/TBST, an HRP-conjugated anti-M13 antibody (AmershamPharmacia Biotech) diluted with TBS or 10 mM (ATP and adenosine)/TBS wasadded to each well. The plate was incubated for one hour. Following washwith TBST or 10 mM (ATP and adenosine)/TBST, the TMB single solution(ZYMED) was added to each well. The chromogenic reaction in the solutionof each well was terminated by adding sulfuric acid. Then, the developedcolor was assessed by measuring absorbance at 450 nm.

Phage ELISA was carried out using 96 isolated clones to obtain from thelibrary of rationally designed antibodies, clone “I6RLSA1-6_(—)011”,which had an antigen-binding activity to human IL-6 in the absence ofATP and adenosine, clone “HSADSA1-6_(—)020”, which had antigen-bindingactivity to human serum albumin (HSA) in the absence of ATP andadenosine, as well as clones “6RRLSA1-6_(—)037” and “6RRLSA1-6_(—)045”,which had an antigen-binding activity to human IL-6 receptor in theabsence of ATP and adenosine (FIGS. 38, 39, and 45).

(16-3) Sequence Analysis of Antibodies for which Adenosine and ATP Serveas a Switch

Genes were amplified using specific primers (SEQ ID NOs: 111 and 112)from clones that had been assessed to have antigen-binding activity inthe absence of adenosine and ATP based on the result of phage ELISAdescribed in (16-2). The nucleotide sequences of the genes wereanalyzed. Based on the analysis result, the amino acid sequences areshown in Table 21 below.

TABLE 21 Heavy chain Light chain Clone name SEQ ID NO SEQ ID NOI6RLSA1-6_011 SEQ ID NO: 95 SEQ ID NO: 96 HSADSA1-6_020 SEQ ID NO: 97SEQ ID NO: 98 6RRLSA1-6_037 SEQ ID NO: 106 SEQ ID NO: 107 6RRLSA1-6_045SEQ ID NO: 108 SEQ ID NO: 109

Example 17 Acquisition of Antibodies that Bind to Antigens in thePresence of Adenosine and ATP from Antibody Library Using MultivalentPhage-Display Technique

(17-1) Acquisition of Antibodies that Bind to Antigens in the Presenceof Small Molecules from Library Using Multivalent Display

Multivalent display of antibodies on phage was used to obtain antibodiesthat have an antigen-binding activity in the presence of adenosineand/or ATP from a phage-display library of rationally designedantibodies. In obtaining antibodies from a library, the acquisitionprobability increases when the ratio of the antigen-binding abilitybetween in the presence and absence of small molecules is greater. Thus,panning based on augmentation of the apparent binding ability wasperformed to efficiently collect antibodies with binding ability in thepresence of small molecules. More specifically, the apparent bindingability was augmented through an avidity effect (effect of multivalentantigen binding) by allowing phages to display antibodies in amultivalent manner. First, a phage-display library of rationallydesigned antibodies was contacted with biotinylated antigens in thepresence of adenosine and ATP to collect a phage-display library ofantibodies that bind to the antigens in the presence of adenosine andATP. Then, according to the method described in Rondot (Nat. Biotechnol.(2001) 19, 75-78), E. coli was infected with the collected phage-displaylibrary of antibodies, and then infected with helper phages that aredeficient in the gene encoding pIII to prepare a multivalentphage-display library of antibodies where antibodies are presented atall pIIIs. The multivalent phage-display library of antibodies wascontacted with biotinylated antigen-streptavidin in the presence ofadenosine and ATP. After the library was collected, phages were elutedfrom the beads in the absence of adenosine and ATP, and collected in theeluate. This cycle of phage preparation and panning was carried outseveral times to screen for antibodies that have antigen-bindingactivity only in the presence of adenosine and/or ATP.

E. coli containing the constructed phage-display phagemid were infectedwith helper phage M13KO7 and cultured overnight at 30° C. to produce amonovalent phage-display library of antibodies. After phage production,2.5 M NaCl/10% PEG was added to the culture medium of E. coli toprecipitate phages. The precipitated phage fraction was diluted with TBSto prepare a phage library solution. Then, BSA was added at a finalconcentration of 4% to the phage library solution. Panning was performedusing antigen-immobilized magnetic beads. The magnetic beads used wereNeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated) andStreptavidin-coated beads (Dynabeads M-280 Streptavidin).

The phage library solution was contacted with an antigen, adenosine, andATP at room temperature for 60 minutes by adding 500 pmol ofbiotin-labeled human IgA-Fc (SEQ ID NO: 99) as the antigen, and a finalconcentration of 1 mM ATP-Na and adenosine to the prepared phage librarysolution. BSA-blocked magnetic beads were added to the phage librarysolution, and the antigen-phage complex was allowed to bind to themagnetic beads at room temperature for 15 minutes. The beads were washedonce with TBS dissolved with ATP and adenosine. Then, 0.5 ml of a 1mg/ml trypsin solution was added to the beads. Immediately after thebeads were suspended at room temperature for 15 minutes, a phagesolution was collected from the isolated beads using a magnetic stand.The collected phage solution was added to 10 ml of E. coli cells ofstrain ER2738 at the logarithmic growth phase (OD600=0.4 to 0.7). The E.coli was incubated with gentle stirring at 37° C. for one hour to beinfected by phage. The infected E. coli was seeded in a 225 mm×225 mmplate. Then, helper phage M13KO7 or M13KO7ApIII (referred to ashyperphage) (PROGEN Biotechnik) was allowed to infect the culture mediumof the seeded E. coli, and phages were collected from the supernatant ofthe culture incubated overnight at 30° C. to prepare a monovalentphage-display antibody library or a multivalent phage-display antibodylibrary solution, respectively.

The first round of panning was carried out to collect phages that arecapable of antigen binding in the presence of adenosine and ATP, whilethe second and subsequent rounds of panning were performed to enrichphages that are capable of antigen binding only in the presence ofadenosine and ATP. Specifically, 250 pmol of the biotin-labeled antigen,and a final concentration of 1 mM adenosine and ATP were each added tothe prepared phage library solution. Thus, the phage library wascontacted with the antigen, adenosine, and ATP at room temperature for60 minutes. BSA-blocked magnetic beads were added, and allowed to bindto the phage-antigen complex for 15 minutes at room temperature. Thebeads were washed with 1 ml of TBST dissolved with adenosine and ATP(hereinafter referred to as (adenosine+ATP)/TBST) and with TBS dissolvedwith adenosine and ATP (hereinafter referred to as (adenosine+ATP)/TBS).Then, the 0.5 ml of TBS was added to the beads. Immediately after thebeads were suspended at room temperature, a phage solution was collectedfrom the isolated beads using a magnetic stand. After this treatment wasrepeated, the two separately eluted phage solutions were combined. ThepIII protein (helper phage-derived protein pIII) that does not displayFab was cleaved off from phages by adding 5 μl of 100 mg/ml trypsin tothe collected phage solution to eliminate the E. coli-infecting abilityof the phages that do not display Fab. The phages collected from thetrypsinized phage solution were added to 10 ml of E. coli cells ofstrain ER2738 at the logarithmic growth phase (OD600=0.4 to 0.7). The E.coli was incubated with gentle stirring at 37° C. for one hour to beinfected with phage. The infected E. coli was seeded in a 225 mm×225 mmplate. Then, in the same manner as used in the first round panning,phages were collected from the culture medium of the seeded E. coli toobtain a monovalent phage-display antibody library and a multivalentphage-display antibody library solution. Panning was performed threetimes to obtain antibodies that have antigen-binding activity in thepresence of adenosine and ATP. Meanwhile, in the third and subsequentrounds of panning, the biotinylated antigen was used at 40 pmol.

(17-2) Assessment of Binding Activity in the Presence of Adenosineand/or ATP by Phage ELISA

Culture supernatants containing phages were collected according to aconventional method (Methods Mol. Biol. (2002) 178, 133-145) from singlecolonies of E. coli obtained by the method described above. Thecollected culture supernatants were ultrafiltrated using NucleoFast 96(MACHERY-NAGEL). 100 μl of the culture supernatants were added to eachwell of NucleoFast 96 and centrifuged (4500 g for 45 minutes) to removeflow-through. After 100 μl of H₂O was added, the NucleoFast 96 waswashed by centrifugation (4500 g for 30 minutes). Finally, 100 μl of TBSwas added, and the NucleoFast 96 was allowed to stand for five minutesat room temperature. A phage solution contained in the supernatant ineach well was collected.

Purified phages, to which TBS or (adenosine+ATP)/TBS was added, weresubjected to ELISA by the following procedure. A StreptaWell 96microtiter plate (Roche) was coated overnight with 100 μl of TBScontaining a biotin-labeled antigen. After the antigen was removed fromeach well of the plate by washing with TBST, the wells were blocked with250 μl of 2% skim milk/TBS for one hour or more. 2% skim milk/TBS wasremoved, and then the prepared, purified phages were added to each well.The plate was allowed to stand for one hour to allow binding ofantibody-displaying phages to the antigen in each well in the presenceor absence of adenosine and ATP. After washing with TBST or(adenosine+ATP)/TBST, an HRP-conjugated anti-M13 antibody (AmershamPharmacia Biotech) diluted with TBS or (adenosine+ATP)/TBS was added toeach well. The plate was incubated for one hour. Following wash withTBST or (adenosine+ATP)/TBST, the TMB single solution (ZYMED) was addedto each well. The chromogenic reaction in the solution of each well wasterminated by adding sulfuric acid. Then, the developed color wasassessed by measuring absorbance at 450 nm. The result shows that agreater number of antibodies that have binding activity in the presenceof small molecules were obtained from the multivalent phage-displayantibody library (FIGS. 40 and 41). This finding suggests thatantibodies that have binding activity in the presence of small moleculescan be obtained more efficiently by using the multivalent phage-displayantibody library method. The result of phage ELISA is shown in Table 22below.

TABLE 22 Monovalent Multivalent presentation presentation Number ofpanning  4  4 Number of clones subjected to ELISA 96 96 Number ofpositive clones (Absorbance > 0.1)  6 28 Number of dependent clones (SM+/− ratio > 1.3)  1 19 Number of dependent clone sequences  1  5(17-3) Assessment of the Binding Ability of Antibodies that UseAdenosine and ATP as a Switch, and Sequence Analysis

Genes were amplified using specific primers (SEQ ID NOs: 111 and 112)from clones that had been assessed to have antigen-binding activity inthe presence of adenosine and ATP based on the result of phage ELISAdescribed in (17-2). The nucleotide sequences of the genes wereanalyzed. As a result, clone “2IADL3C5-4_(—)048 (heavy chain, SEQ IDNOs: 100; light chain, SEQ ID NO: 101)” which exhibits antigen-bindingactivity in the presence of adenosine and ATP was obtained (FIG. 42).

Example 18 Characterization of ATP/Adenosine-Dependent AntibodiesObtained from Library

(18-1) Preparation of ATP/Adenosine-Dependent Antibodies Obtained fromLibrary

Genes were amplified using specific primers from clones6RAD2C1-4_(—)001, 6RAD2C1-4_(—)005, 6RAD2C1-4_(—)011, 6RAD2C1-4_(—)026,6RAD2C1-4_(—)030, 6RAD2C1-4_(—)042, 6RAD2C1-4_(—)076, 6RDL3C1-4_(—)085,and 6RDL3C5-4_(—)011, which were obtained as described in Example 10 andwere assessed to have binding activity to biotin-labeled hIL-6R (hI-L6)in the presence of ATP or adenosine; and their nucleotide sequences wereanalyzed (Table 23).

TABLE 23 Heavy chain Light chain Clone name SEQ ID NO SEQ ID NO6RAD2C1-4_001 SEQ ID NO: 113 SEQ ID NO: 114 6RAD2C1-4_005 SEQ ID NO: 115SEQ ID NO: 116 6RAD2C1-4_011 SEQ ID NO: 82 SEQ ID NO: 83 6RAD2C1-4_026SEQ ID NO: 117 SEQ ID NO: 118 6RAD2C1-4_030 SEQ ID NO: 119 SEQ ID NO:120 6RAD2C1-4_042 SEQ ID NO: 121 SEQ ID NO: 122 6RAD2C1-4_076 SEQ ID NO:84 SEQ ID NO: 85 6RDL3C1-4_085 SEQ ID NO: 123 SEQ ID NO: 1246RDL3C5-4_011 SEQ ID NO: 125 SEQ ID NO: 126

The variable regions of 6RAD2C1-4_(—)001, 6RAD2C1-4_(—)005,6RAD2C1-4_(—)011, 6RAD2C1-4_(—)026, 6RAD2C1-4_(—)030, 6RAD2C1-4_(—)042,6RAD2C1-4_(—)076, 6RDL3C1-4_(—)085, and 6RDL3C5-4_(—)011 were insertedinto an animal expression plasmid for human IgG1/Kappa that has theantibody heavy chain constant region of SEQ ID NO: 90 and the lightchain kappa constant region sequence of SEQ ID NO: 91. Antibodies wereexpressed using the method described below. FreeStyle 293-F (Invitrogen)which was derived from human fetal kidney cells were suspended at a celldensity of 1.33×10⁶ cells/ml in the FreeStyle 293 Expression Medium(Invitrogen), and aliquoted at 3 ml into each well of a 6-well plate.The plasmid DNA was transfected into the cells by lipofection. From theculture supernatants after four days of culture in a CO₂ incubator (37°C., 8% CO₂, 90 rpm), antibodies were purified by a method known to thoseskilled in the art using rProtein A Sepharose™ Fast Flow (AmershamBiosciences). Absorbance of the purified antibody solutions was measuredat 280 nm using a spectrophotometer. From values obtained by themeasurement, concentrations of purified antibodies were calculated usingan extinction coefficient determined by the PACE method (Protein Science(1995) 4, 2411-2423).

(18-2) Assessment of the Effect of Various Small Molecules on Human IL6Receptor Binding by Surface Plasmon Resonance

Biacore T200 (GE Healthcare) was used to evaluate the effect of varioussmall molecules on the antigen-antibody reaction of IL-6R with 9ATP/adenosine-dependent antibodies (6RAD2C1-4_(—)001, 6RAD2C1-4_(—)005,6RAD2C1-4_(—)011, 6RAD2C1-4_(—)026, 6RAD2C1-4_(—)030, 6RAD2C1-4_(—)042,6RAD2C1-4_(—)076, 6RDL3C1-4_(—)085, and 6RDL3C5-4_(—)011) obtained fromthe library. The running buffer was used: 20 mmol/l ACES, 150 mmol/lNaCl, 0.05% (w/v) Tween20, pH 7.4. Assay was carried out at 25° C. IL-6Rwas immobilized onto sensor chip CM5 by amine coupling; and theantibodies were allowed to interact as analyte for 120 seconds, andchanges in the amount of binding were observed. For dilution of theantibodies, the running buffer or the running buffer containing any oneof ATP, ADP, AMP, cAMP, and adenosine (ADO) were used. The finalconcentration of each small molecule and the final concentration of eachantibody were adjusted to 1 mM and 1 μM, respectively. Meanwhile, underthe 1 mM ATP condition, assay was carried out with a series of stepwiseantibody concentrations. The dissociation constant K_(D) (mol/L) of eachclone for IL-6R was calculated from a plot of equilibrium value againstantibody concentration. The parameters were calculated using the BiacoreT200 Evaluation Software (GE Healthcare). The dissociation constantK_(D) of each clone in the presence of 1 mM ATP is shown in Table 24.

TABLE 24 Dissociation constant K_(D) Clone name (mol/L) 6RAD2C1-4_013.0E−07 6RAD2C1-4_05 3.4E−07 6RAD2C1-4_11 2.3E−07 6RAD2C1-4_26 2.1E−076RAD2C1-4_30 3.3E−07 6RAD2C1-4_42 2.5E−07 6RAD2C1-4_76 2.5E−076RDL3C1-4_85 3.9E−07 6RDL3C5-4_11 1.3E−07

The amount of each clone that binds to IL-6R as determined by this assayin the presence or absence of 1 mM small molecules is shown in FIG. 43.As shown in FIG. 43, each clone binds to IL-6R in the presence of 1 mMATP, but their IL-6R binding was not detectable in the absence of ATP.This demonstrates that each clone has the property that it binds toIL-6R via ATP as a switch. In small molecules besides ATP, binding ofall clones was observed in the presence of ADP, and some clones wereshown to bind in the presence of AMP and cAMP.

IL-6R Binding was not Detectable in the Presence of ADO.

This demonstrates that antibodies that bind to target antigens in thepresence of any one or more of ATP, ADP, AMP, and cAMP can be obtainedby using rationally designed libraries. As described in this Example,panning was carried out in the presence of both ATP and ADO which bindto antibody ATNLSA1-4_D12 used as a reference in designing the designlibraries. The result showed that antibodies that strongly bind totarget antigens were isolated in the presence of ATP which stronglybinds to ATNLSA1-4_D12 but not in the presence of ADO which binds moreweakly to ATNLSA1-4_D12 than ATP. Antibodies that bind to antigens in amanner depending on a desired small molecule alone can be obtained byisolating antigen-binding antibodies by contacting antigens withlibraries in the presence of the small molecule alone. For example,antibodies that bind in the presence of ADO can be efficiently obtainedfrom libraries by performing panning in the presence of ADO alone.

(18-3) Effect of ATP on the ADCC Activity of Obtained Antibodies

The obtained antibodies 6RAD2C1-4_(—)030 and 6RAD2C1-4_(—)011 wereassessed by the method described below to test whether ADCC activityagainst hIL-6R-expressing cells is mediated via hIL-6R binding of theantibodies in the presence of adenosine triphosphate (ATP). Thisassessment was carried out using variant 6RAD2C1-4_(—)030 with increasedeffector activity (antibody heavy chain variable region, SEQ ID NO: 119;antibody light chain variable region, SEQ ID NO: 120; antibody heavychain constant region, SEQ ID NO: 90, antibody light chain constantregion, SEQ ID NO: 91) and variant 6RAD2C1-4_(—)011 with increasedeffector activity (antibody heavy chain variable region, SEQ ID NO: 82;antibody light chain variable region, SEQ ID NO: 83; antibody heavychain constant region, SEQ ID NO: 90; antibody light chain constantregion, SEQ ID NO: 91) prepared as described in Example 18-1, as well asa known anti-human IL-6R antibody MRA (antibody heavy chain variableregion, SEQ ID NO: 92; antibody light chain variable region, SEQ ID NO:92; antibody heavy chain constant region, SEQ ID NO: 90; antibody lightchain constant region, SEQ ID NO: 91) prepared as described in Example15-5. At various antibody concentrations in the presence or absence ofATP, 6RAD2C1-4_(—)030, 6RAD2C1-4_(—)011, and MRA were assessed for theADCC activity to hIL-6R-expressing cells according to the methoddescribed in Reference Example 3. The assay result is shown in FIG. 44.

The assay result confirmed antibody concentration-dependent ADCCactivity by 6RAD2C1-4_(—)030 and 6RAD2C1-4_(—)011 in the presence ofATP. This finding shows that ADCC activity is induced againstantigen-expressing cells by antigen-antibody binding mediated by notonly kynurenine but also ATP; and thus it is revealed that forantibodies that bind to antigens in the presence of small molecules as aswitch, their function of antitumor activity such as ADCC activity canalso be regulated by the presence of small molecules serving as aswitch.

These results suggest strong induction of ADCC activity againsthIL-6R-expressing cells is expected in tumor tissues where ATPconcentration is high, and weak induction of ADCC activity in normaltissues where ATP concentration is low. Based on the above, byadministering antibodies for which ATP serves as a switch, thecytotoxicity against normal tissues expressing a target antigen can bereduced without impairing the pharmaceutical effect on tumor tissuesexpressing the target antigen.

Reference Example 1 ADCC Activity of Test Antibodies Using HumanPeripheral Blood Mononuclear Cells as Effector Cells

Antibodies that bind antigens in a kynurenine-dependent manner wereassessed for their ADCC activity against antigen-expressing cells atdifferent antibody concentrations according to the method describedbelow. Human peripheral blood mononuclear cells (hereinafter referred toas human PBMC) were used as effector cells to measure the ADCC activityof each test antibody as follows.

(1) Preparation of Human PBMC Solution

Syringes pre-filled with 200 μl of 1000 units/ml heparin solution(Novo-Heparin 5000 units for Injection; Novo Nordisk) were used tocollect 50 ml of peripheral blood from healthy volunteers (male adult)affiliated with Chugai Pharmaceutical Co. Ltd. The peripheral blood wasdiluted two-fold with PBS(−), and divided into four equal parts, each ofwhich was transferred into a pre-centrifuged leukocyte separation tubeLeucosep (Greiner Bio-One) containing 15 ml of Ficoll-Paque PLUS. Theseparation tubes containing an aliquot of the peripheral blood werecentrifuged at 2150 rpm for ten minutes at room temperature. Then, theresulting mononuclear cell fractions were collected from the tubes. Thecells in each fraction were washed once with RPMI-1640 (nacalai tesque)supplemented with 10% FBS (hereinafter referred to as 10% FBS/RPMI), andthen suspended at a cell density of 1×10⁷ cells/ml in 10% FBS/RPMI. Thecell suspensions were used as the human PBMC solution in subsequentexperiments.

(2) Preparation of Target Cells

0.74 MBq of Cr-51 was added to 3×10⁶ cells of BaF/hIL6R (Mihara et al.,(Int. Immunopharmacol. (2005) 5, 1731-40) which is Ba/F3 cellsexpressing human IL-6 receptor. Then, the cells were incubated in 5% CO₂incubator at 37° C. for 1 hour. After washing 3 times with 10% FBS/RPMI,the cells were suspended at a cell density of 2×10⁵ cells/ml in 10%FBS/RPMI. The cell suspension was used as the target cells in subsequentexperiments.

(3) Preparation of Kynurenine Solution

L-Kynurenine (sigma) was diluted to 5 mM with PBS(−), and then itsconcentration was adjusted to 400 μM using 10% FBS/RPMI. The solutionwas used as the kynurenine solution in subsequent experiments.

(4) Chrome Release Assay (ADCC)

ADCC activity was assessed based on specific chrome release ratedetermined by chrome release assay. First, antibody solutions preparedat various concentrations (0, 0.04, 0.4, 4, and 40 μg/ml) were added at50 μl to each well of a round-bottomed 96-well plate. Then, the targetcells prepared as described in (2) were seeded at 50 μl (1×10⁴cells/well) to the wells. Furthermore, the kynurenine solution preparedas described in (3) was added at 50 μl to the wells, and the plate wasallowed to stand at room temperature for 15 minutes. Then, the humanPBMC solution prepared as described in (1) was added at 50 μl to eachwell (5×10⁵ cells/well). The plate was allowed to stand in5% CO₂incubator at 37° C. for 4 hours, followed by centrifugation. 100 μl ofculture supernatant from each well of the plate was measured forradioactivity using a gamma counter. The specific chrome release ratewas determined based on the equation below.

Chrome release rate (%)=(A−C)×100/(B−C)

In this equation, “A” represents mean radioactivity (cpm) of 100 μl ofculture supernatant in each well. “B” represents mean radioactivity(cpm) of 100 μl of culture supernatant in a well containing targetcells, 50 μl of 4% NP-40 aqueous solution (Nonidet P-40; NacalaiTesques), and 100 μl of 10% FBS/RPMI. Furthermore, “C” represents meanradioactivity (cpm) of 100 μl of culture supernatant in a wellcontaining target cells, 150 μl of 10% FBS/RPMI or 100 μl of 10%FBS/RPMI, and 50 μl of kynurenine solution. The test was carried out induplicate. The mean specific chrome release rate (%) that reflects theADCC activity of each test antibody was calculated based on the assaydescribed above.

Reference Example 2 ADCC Activity of Each Test Antibody Using HumanPeripheral Blood Mononuclear Cells as Effector Cells

Antibodies that bind to antigens in a kynurenine-dependent manner wereassessed for their ADCC activity against antigen-expressing cells atvarious kynurenine concentrations according to the method describedbelow. Using human peripheral blood mononuclear cells as effector cells,the ADCC activity of each test antibody was assayed as follows. HumanPBMC solution and target cells were prepared by the same method asdescribed in Reference Example 1.

(1) Preparation of Kynurenine Solutions

L-Kynurenine (sigma) was diluted to 5 mM with PBS(−), and then itsconcentration was adjusted to 1200, 400, 133, 44, 14.8, and 4.9 μM using10% FBS/RPMI. The solutions were used as kynurenine solutions insubsequent experiments.

(2) Chrome Release Assay (ADCC)

ADCC activity was assessed based on specific chrome release ratedetermined by chrome release assay. First, an antibody solution preparedto 200 μg/ml was added at 50 μl to each well of a round-bottomed 96-wellplate. Then, the target cells prepared as described above were seeded at50 μl (1×10⁴ cells/well) to the wells. Furthermore, the kynureninesolution prepared at each concentration as described in (1) was added at50 μl to the wells, and the plate was allowed to stand at roomtemperature for 15 minutes. Then, the human PBMC solution prepared asdescribed above was added at 50 μl to each well (5×10⁵ cells/well). Theplate was allowed to stand in 5% CO₂ incubator at 37° C. for 4 hours,followed by centrifugation. 100 μl of culture supernatant from each wellof the plate was measured for radioactivity using a gamma counter. Thespecific chrome release rate was determined based on the equationdescribed in Reference Example 1.

Reference Example 3 ADCC Activity of Test Antibodies Using Human NK CellLine NK92 as Effector Cells

Antibodies that bind to antigens in an ATP-dependent manner wereassessed for their ADCC activity against antigen-expressing cells atvarious antibody concentrations according to the method described below.The ADCC activity of each test antibody was assayed by using as effectorcells NK92-CD16(V) resulting from forced expression of human FcgRIIIa inhuman NK cell line NK92 as follows.

(1) Preparation of NK92-CD16(V)

NK92-CD16(V) was suspended at a cell density of 1×10⁷ cells/ml inRPMI/10% FBS. The cell suspension was used as an NK92-CD16(V) solutionin subsequent experiments.

(2) Preparation of Target Cells

0.74 MBq of Cr-51 was added to 3×10⁶ cells of CHO/hIL6R which is CHOcells expressing human IL-6 receptor. Then, the cells were incubated in5% CO₂ incubator at 37° C. for 1 hour. After washing 3 times with 10%FBS/RPMI, the cells were suspended at a cell density of 2×10⁵ cells/mlin 10% FBS/RPMI. The cell suspension was used as target cells insubsequent experiments.

(3) Preparation of ATP Solution

ATP (sigma) was diluted to 100 mM with 10% FBS/RPMI, and then itsconcentration was adjusted to 4 mM. The solution was used as an ATPsolution in subsequent experiments.

(4) Chrome Release Assay (ADCC)

ADCC activity was assessed based on the specific chrome release ratedetermined by chrome release assay. First, antibody solutions preparedto various concentrations (0, 0.04, 0.4, 4, or 40 μg/ml) were each addedat 50 μl to a well of a round-bottomed 96-well plate. Then, the targetcells prepared as described in (2) were seeded at 50 μl (1×10⁴cells/well) to the wells. Furthermore, the ATP solution prepared asdescribed in (3) was added at 50 μl to the wells, and the plate wasallowed to stand at room temperature for 15 minutes. Then, theNK92-CD16(V) solution prepared as described in (1) was added at 50 μl toeach well (5×10⁵ cells/well). The plate was allowed to stand in 5% CO₂incubator at 37° C. for 4 hours, followed by centrifugation. 100 μl ofculture supernatant from each well of the plate was measured forradioactivity using a gamma counter. The specific chrome release ratewas determined based on the equation described in Reference Example 1.

1. An antigen-binding molecule comprising an antigen-binding domainwhose antigen-binding activity varies depending on the concentration ofa target tissue-specific compound.
 2. The antigen-binding molecule ofclaim 1, wherein the target tissue is a cancer tissue.
 3. Theantigen-binding molecule of claim 2, wherein the compound specific to acancer tissue is a metabolite specific to a cancer cell, a metabolitespecific to an immune cell that has infiltrated into a cancer tissue, ora metabolite specific to a stromal cell in a cancer tissue.
 4. Theantigen-binding molecule of claim 1, wherein the target tissue is aninflamed tissue.
 5. The antigen-binding molecule of claim 4, wherein thecompound specific to an inflamed tissue is a metabolite specific to animmune cell that has infiltrated into an inflamed tissue or a metabolitespecific to a normal cell that has been damaged in an inflamed tissue.6. The antigen-binding molecule of claim 1, wherein the compound is atleast one compound selected from a nucleoside having a purine ringstructure, an amino acid and its metabolite, a lipid and its metabolite,a primary metabolite of glycometabolism, and nicotinamide and itsmetabolite.
 7. The antigen-binding molecule of claim 6, wherein thecompound is at least one compound selected from adenosine, adenosinetriphosphate, inosine, alanine, glutamic acid, aspartic acid,kynurenine, prostaglandin E2, succinic acid, citric acid, and1-methylnicotinamide.
 8. The antigen-binding molecule of any one ofclaims 1 to 7, wherein the antigen is a membrane-type molecule.
 9. Theantigen-binding molecule of any one of claims 1 to 8, which is anantigen-binding molecule that has a neutralizing activity.
 10. Theantigen-binding molecule of any one of claims 1 to 9, which is anantigen-binding molecule that has a cytotoxic activity.
 11. Theantigen-binding molecule of any one of claims 1 to 10, which comprisesan Fc region.
 12. The antigen-binding molecule of claim 11, wherein theFc region is an Fc region contained in the constant region of SEQ IDNOs: 5, 6, 7, or
 8. 13. The antigen-binding molecule of claim 11,wherein the Fc region comprises an altered FcγR-binding Fc region thathas a higher Fcγ receptor-binding activity than the Fcγ receptor-bindingactivity of a native human IgG Fc region.
 14. The antigen-bindingmolecule of claim 13, wherein at least one or more amino acids selectedfrom the group consisting of amino acids at positions 221, 222, 223,224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258,260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305,311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329,330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380,382, 385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 according toEU numbering, in the amino acid sequence of the altered FcγR-binding Fcregion are different from the amino acids of the native human IgG Fcregion.
 15. The antigen-binding molecule of claim 14, which comprises atleast one or more amino acids selected from the group consisting of: Lysor Tyr for the amino acid at position 221; Phe, Trp, Glu, or Tyr for theamino acid at position 222; Phe, Trp, Glu, or Lys for the amino acid atposition 223; Phe, Trp, Glu, or Tyr for the amino acid at position 224;Glu, Lys, or Trp for the amino acid at position 225; Glu, Gly, Lys, orTyr for the amino acid at position 227; Glu, Gly, Lys, or Tyr for theamino acid at position 228; Ala, Glu, Gly, or Tyr for the amino acid atposition 230; Glu, Gly, Lys, Pro, or Tyr for the amino acid at position231; Glu, Gly, Lys, or Tyr for the amino acid at position 232; Ala, Asp,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, orTyr for the amino acid at position 233; Ala, Asp, Glu, Phe, Gly, His,Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for theamino acid at position 234; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met,Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid atposition 235; Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro,Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position 236;Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp, or Tyr for the amino acid at position 237; Asp, Glu, Phe, Gly,His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr forthe amino acid at position 238; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr for the amino acid atposition 239; Ala, Ile, Met, or Thr for the amino acid at position 240;Asp, Glu, Leu, Arg, Trp, or Tyr for the amino acid at position 241; Leu,Glu, Leu, Gln, Arg, Trp, or Tyr for the amino acid at position 243; Hisfor the amino acid at position 244; Ala for the amino acid at position245; Asp, Glu, His, or Tyr for the amino acid at position 246; Ala, Phe,Gly, His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acid at position247; Glu, His, Gln, or Tyr for the amino acid at position 249; Glu orGln for the amino acid at position 250; Phe for the amino acid atposition 251; Phe, Met, or Tyr for the amino acid at position 254; Glu,Leu, or Tyr for the amino acid at position 255; Ala, Met, or Pro for theamino acid at position 256; Asp, Glu, His, Ser, or Tyr for the aminoacid at position 258; Asp, Glu, His, or Tyr for the amino acid atposition 260; Ala, Glu, Phe, Ile, or Thr for the amino acid at position262; Ala, Ile, Met, or Thr for the amino acid at position 263; Asp, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, orTyr for the amino acid at position 264; Ala, Leu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for theamino acid at position 265; Ala, Ile, Met, or Thr for the amino acid atposition 266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Thr, Val, Trp, or Tyr for the amino acid at position 267; Asp, Glu,Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp for theamino acid at position 268; Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position 269; Glu,Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr forthe amino acid at position 270; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acidat position 271; Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 272; Phe or Ile forthe amino acid at position 273; Asp, Glu, Phe, Gly, His, Ile, Leu, Met,Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position274; Leu or Trp for the amino acid at position 275; Asp, Glu, Phe, Gly,His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the aminoacid at position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,Gln, Arg, Ser, Thr, Val, or Trp for the amino acid at position 278; Alafor the amino acid at position 279; Ala, Gly, His, Lys, Leu, Pro, Gln,Trp, or Tyr for the amino acid at position 280; Asp, Lys, Pro, or Tyrfor the amino acid at position 281; Glu, Gly, Lys, Pro, or Tyr for theamino acid at position 282; Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg,or Tyr for the amino acid at position 283; Asp, Glu, Leu, Asn, Thr, orTyr for the amino acid at position 284; Asp, Glu, Lys, Gln, Trp, or Tyrfor the amino acid at position 285; Glu, Gly, Pro, or Tyr for the aminoacid at position 286; Asn, Asp, Glu, or Tyr for the amino acid atposition 288; Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for theamino acid at position 290; Asp, Glu, Gly, His, Ile, Gln, or Thr for theamino acid at position 291; Ala, Asp, Glu, Pro, Thr, or Tyr for theamino acid at position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,Ser, Thr, Val, Trp, or Tyr for the amino acid at position 293; Phe, Gly,His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr forthe amino acid at position 294; Asp, Glu, Phe, Gly, His, Ile, Lys, Met,Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position295; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr, or Val for the amino acid at position 296; Asp, Glu, Phe, Gly, His,Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for theamino acid at position 297; Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn,Gln, Arg, Thr, Val, Trp, or Tyr for the amino acid at position 298; Ala,Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,Val, Trp, or Tyr for the amino acid at position 299; Ala, Asp, Glu, Gly,His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp forthe amino acid at position 300; Asp, Glu, His, or Tyr for the amino acidat position 301; Ile for the amino acid at position 302; Asp, Gly, orTyr for the amino acid at position 303; Asp, His, Leu, Asn, or Thr forthe amino acid at position 304; Glu, Ile, Thr, or Tyr for the amino acidat position 305; Ala, Asp, Asn, Thr, Val, or Tyr for the amino acid atposition 311; Phe for the amino acid at position 313; Leu for the aminoacid at position 315; Glu, or Gln for the amino acid at position 317;His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr for the amino acid atposition 318; Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val,Trp, or Tyr for the amino acid at position 320; Ala, Asp, Phe, Gly, His,Ile, Pro, Ser, Thr, Val, Trp, or Tyr for the amino acid at position 322;Ile for the amino acid at position 323; Asp, Phe, Gly, His, Ile, Leu,Met, Pro, Arg, Thr, Val, Trp, or Tyr for the amino acid at position 324;Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 325; Ala, Asp, Glu,Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr for theamino acid at position 326; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr for the amino acid at position327; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp, or Tyr for the amino acid at position 328; Asp, Glu,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, orTyr for the amino acid at position 329; Cys, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the aminoacid at position 330; Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val,Trp, or Tyr for the amino acid at position 331; Ala, Asp, Glu, Phe, Gly,His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr forthe amino acid at position 332; Ala, Asp, Glu, Phe, Gly, His, Ile, Leu,Met, Pro, Ser, Thr, Val, or Tyr for the amino acid at position 333; Ala,Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid at position 334; Asp,Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr forthe amino acid at position 335; Glu, Lys, or Tyr for the amino acid atposition 336; Glu, His, or Asn for the amino acid at position 337; Asp,Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr for the amino acidat position 339; Ala or Val for the amino acid at position 376; Gly orLys for the amino acid at position 377; Asp for the amino acid atposition 378; Asn for the amino acid at position 379; Ala, Asn, or Serfor the amino acid at position 380; Ala, or Ile for the amino acid atposition 382; Glu for the amino acid at position 385; Thr for the aminoacid at position 392; Leu for the amino acid at position 396; Lys forthe amino acid at position 421; Asn for the amino acid at position 427;Phe, or Leu for the amino acid at position 428; Met for the amino acidat position 429; Trp for the amino acid at position 434; Ile for theamino acid at position 436; and Gly, His, Ile, Leu, or Tyr for the aminoacid at position 440 according to EU numbering in the amino acidsequence of the altered FcγR-binding Fc region.
 16. The antigen-bindingmolecule of claim 11, wherein the Fc region is modified so that there isa higher proportion of Fc region bound by a fucose-deficient sugar chainin a composition of sugar chain bound at position 297, according to EUnumbering, of the Fc region, or so that there is a higher proportion ofFc region with an added bisecting N-acetylglucosamine.
 17. Theantigen-binding molecule of any one of claims 11 and 13 to 16, whereinthe FcRn-binding activity of the Fc region under an acidic pH rangecondition is enhanced compared to the FcRn-binding activity of the Fcregion of SEQ ID NO: 5, 6, 7, or
 8. 18. The antigen-binding molecule ofclaim 17, wherein the Fc region is an Fc region with substitution of atleast one or more amino acids selected from the group consisting ofamino acids at positions 238, 244, 245, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285, 286, 288,293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317, 318, 332, 339,340, 341, 343, 356, 360, 362, 375, 376, 377, 378, 380, 382, 385, 386,387, 388, 389, 400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434,435, 436, 438, 439, 440, 442, and 447, according to EU numbering, in theamino acid sequence of the Fc region comprised in the constant region ofSEQ ID NO: 5, 6, 7, or
 8. 19. The antigen-binding molecule of claim 18,wherein the Fc region comprises at least one or more amino acidsselected from the group consisting of: Leu for the amino acid atposition 238; Leu for the amino acid at position 244; Arg for the aminoacid at position 245; Pro for the amino acid at position 249; Gln or Glufor the amino acid at position 250; Arg, Asp, Glu, or Leu for the aminoacid at position 251; Phe, Ser, Thr, or Tyr for the amino acid atposition 252; Ser or Thr for the amino acid at position 254; Arg, Gly,Ile, or Leu for the amino acid at position 255; Ala, Arg, Asn, Asp, Gln,Glu, Pro, or Thr for the amino acid at position 256; Ala, Ile, Met, Asn,Ser, or Val for the amino acid at position 257; Asp for the amino acidat position 258; Ser for the amino acid at position 260; Leu for theamino acid at position 262; Lys for the amino acid at position 270; Leu,or Arg for the amino acid at position 272; Ala, Asp, Gly, His, Met, Asn,Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid at position 279; Ala,Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, orTyr for the amino acid at position 283; Asn for the amino acid atposition 285; Phe for the amino acid at position 286; Asn or Pro for theamino acid at position 288; Val for the amino acid at position 293, Ala,Glu, Gln, or Met for the amino acid at position 307; Ala, Glu, Ile, Lys,Leu, Met, Ser, Val, or Trp for the amino acid at position 311; Pro forthe amino acid at position 309; Ala, Asp, or Pro for the amino acid atposition 312; Ala or Leu for the amino acid at position 314; Lys for theamino acid at position 316; Pro for the amino acid at position 317; Asnor Thr for the amino acid at position 318; Phe, His, Lys, Leu, Met, Arg,Ser, or Trp for the amino acid at position 332; Asn, Thr, or Trp for theamino acid at position 339; Pro for the amino acid at position 341; Glu,His, Lys, Gln, Arg, Thr, or Tyr for the amino acid at position 343; Argfor the amino acid at position 375; Gly, Ile, Met, Pro, Thr, or Val forthe amino acid at position 376; Lys for the amino acid at position 377;Asp, Asn, or Val for the amino acid at position 378; Ala, Asn, Ser, orThr for the amino acid at position 380; Phe, His, Ile, Lys, Leu, Met,Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid at position382; Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the amino acid atposition 385; Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr for the aminoacid at position 386; Ala, Arg, His, Pro, Ser, or Thr for the amino acidat position 387; Asn, Pro, or Ser for the amino acid at position 389;Asn for the amino acid at position 423; Asn for the amino acid atposition 427; Leu, Met, Phe, Ser, or Thr for the amino acid at position428; Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, or Tyr for the amino acid at position 430; His or Asn for the aminoacid at position 431; Arg, Gln, His, Ile, Lys, Pro, or Ser for the aminoacid at position 433; Ala, Gly, His, Phe, Ser, Trp, or Tyr for the aminoacid at position 434; Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for theamino acid at position 436; Lys, Leu, Thr, or Trp for the amino acid atposition 438; Lys for the amino acid at position 440; Lys for the aminoacid at position 442; and Ile, Pro, or Thr for the amino acid atposition 308; as indicated by EU numbering, in the amino acid sequenceof the Fc region comprised in the constant region of SEQ ID NO: 5, 6, 7,or
 8. 20. The antigen-binding molecule of any one of claims 1 to 19,wherein the antigen-binding domain is a multispecific or amultiparatopic antigen-binding domain.
 21. The antigen-binding moleculeof claim 20, wherein an antigen bound by at least one of theantigen-binding domains is a membrane-type molecule expressed on acancer cell membrane, and an antigen bound by at least one of theantigen-binding domains is a membrane-type molecule expressed on aneffector cell membrane.
 22. The antigen-binding molecule of claim 21,wherein the effector cell is an NK cell, a macrophage, or a T cell. 23.The antigen-binding molecule of claim 21 or 22, wherein themembrane-type molecule expressed on an effector cell membrane is aTCR-constituting polypeptide, CD2, CD3, CD28, CD44, CD16, CD32, CD64, orNKG2D.
 24. The antigen-binding molecule of claim 20, wherein an antigenbound by at least one of the antigen-binding domains is a membrane-typemolecule expressed on a cancer cell membrane, and an antigen bound by atleast one of the antigen-binding domains is a cytotoxic substance. 25.The antigen-binding molecule of any one of claims 20 to 24, wherein theantigen-binding molecule is an antibody fragment.
 26. Theantigen-binding molecule of any one of claims 1 to 24, wherein theantigen-binding molecule is an antibody.
 27. The antigen-bindingmolecule of any one of claims 1 to 7, wherein the antigen is a solublemolecule.
 28. The antigen-binding molecule of claim 27, which is anantigen-binding molecule having a neutralizing activity.
 29. Theantigen-binding molecule of claim 27 or 28, which comprises an Fcregion.
 30. The antigen-binding molecule of claim 29, wherein the Fcregion is an Fc region comprised in the constant region of SEQ ID NO: 5,6, 7, or
 8. 31. The antigen-binding molecule of claim 29, wherein theFcRn-binding activity of the Fc region under an acidic pH rangecondition is enhanced compared to the FcRn-binding activity of the Fcregion comprised in the constant region of SEQ ID NO: 5, 6, 7, or
 8. 32.The antigen-binding molecule of claim 31, wherein the Fc region is an Fcregion with substitution of at least one or more amino acids selectedfrom the group consisting of amino acids at positions 238, 244, 245,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262, 265, 270,272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312,314, 316, 317, 318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376,377, 378, 380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424,427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447,according to EU numbering, in the amino acid sequence of the Fc regioncomprised in the constant region of SEQ ID NO: 5, 6, 7, or
 8. 33. Theantigen-binding molecule of claim 32, wherein the Fc region comprises atleast one or more amino acids selected from the group consisting of: Leufor the amino acid at position 238; Leu for the amino acid at position244; Arg for the amino acid at position 245; Pro for the amino acid atposition 249; Gln or Glu for the amino acid at position 250; Arg, Asp,Glu, or Leu for the amino acid at position 251; Phe, Ser, Thr, or Tyrfor the amino acid at position 252; Ser or Thr for the amino acid atposition 254; Arg, Gly, Ile, or Leu for the amino acid at position 255;Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr for the amino acid at position256; Ala, Ile, Met, Asn, Ser, or Val for the amino acid at position 257;Asp for the amino acid at position 258; Ser for the amino acid atposition 260; Leu for the amino acid at position 262; Lys for the aminoacid at position 270; Leu, or Arg for the amino acid at position 272;Ala, Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for theamino acid at position 279; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn,Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid at position 283;Asn for the amino acid at position 285; Phe for the amino acid atposition 286; Asn or Pro for the amino acid at position 288; Val for theamino acid at position 293, Ala, Glu, Gln, or Met for the amino acid atposition 307; Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp for theamino acid at position 311; Pro for the amino acid at position 309; Ala,Asp, or Pro for the amino acid at position 312; Ala or Leu for the aminoacid at position 314; Lys for the amino acid at position 316; Pro forthe amino acid at position 317; Asn or Thr for the amino acid atposition 318; Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the aminoacid at position 332; Asn, Thr, or Trp for the amino acid at position339; Pro for the amino acid at position 341; Glu, His, Lys, Gln, Arg,Thr, or Tyr for the amino acid at position 343; Arg for the amino acidat position 375; Gly, Ile, Met, Pro, Thr, or Val for the amino acid atposition 376; Lys for the amino acid at position 377; Asp, Asn, or Valfor the amino acid at position 378; Ala, Asn, Ser, or Thr for the aminoacid at position 380; Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr, Val, Trp, or Tyr for the amino acid at position 382; Ala, Arg, Asp,Gly, His, Lys, Ser, or Thr for the amino acid at position 385; Arg, Asp,Ile, Lys, Met, Pro, Ser, or Thr for the amino acid at position 386; Ala,Arg, His, Pro, Ser, or Thr for the amino acid at position 387; Asn, Pro,or Ser for the amino acid at position 389; Asn for the amino acid atposition 423; Asn for the amino acid at position 427; Leu, Met, Phe,Ser, or Thr for the amino acid at position 428; Ala, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr for the amino acidat position 430; His or Asn for the amino acid at position 431; Arg,Gln, His, Ile, Lys, Pro, or Ser for the amino acid at position 433; Ala,Gly, His, Phe, Ser, Trp, or Tyr for the amino acid at position 434; Arg,Asn, His, Ile, Leu, Lys, Met, or Thr for the amino acid at position 436;Lys, Leu, Thr, or Trp for the amino acid at position 438; Lys for theamino acid at position 440; Lys for the amino acid at position 442; andIle, Pro, or Thr for the amino acid at position 308; as indicated by EUnumbering, in the amino acid sequence of the Fc region comprised in theconstant region of SEQ ID NO: 5, 6, 7, or
 8. 34. The antigen-bindingmolecule of claim 29, wherein the FcRn-binding activity of the Fc regionunder a neutral pH range condition is enhanced compared to theFcRn-binding activity of the Fc region comprised in the constant regionof SEQ ID NO: 5, 6, 7, or
 8. 35. The antigen-binding molecule of claim34, wherein the Fc region is an Fc region with substitution of at leastone or more amino acids selected from the group consisting of aminoacids at positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265,286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317,332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428, 433,434, and 436 according to EU numbering, in the amino acid sequence ofthe Fc region comprised in the constant region of SEQ ID NO: 5, 6, 7, or8.
 36. The antigen-binding molecule of claim 35, wherein the Fc regioncomprises at least one or more amino acids selected from the groupconsisting of: Met for the amino acid at position 237; Ile for the aminoacid at position 248; Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr forthe amino acid at position 250; Phe, Trp, or Tyr for the amino acid atposition 252; Thr for the amino acid at position 254; Glu for the aminoacid at position 255; Asp, Asn, Glu, or Gln for the amino acid atposition 256; Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val for theamino acid at position 257; His for the amino acid at position 258; Alafor the amino acid at position 265; Ala or Glu for the amino acid atposition 286; His for the amino acid at position 289; Ala for the aminoacid at position 297; Ala for the amino acid at position 303; Ala forthe amino acid at position 305; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr for the amino acid atposition 307; Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr for the aminoacid at position 308; Ala, Asp, Glu, Pro, or Arg for the amino acid atposition 309; Ala, His, or Ile for the amino acid at position 311; Alaor His for the amino acid at position 312; Lys or Arg for the amino acidat position 314; Ala, Asp, or His for the amino acid at position 315;Ala for the amino acid at position 317; Val for the amino acid atposition 332; Leu for the amino acid at position 334; His for the aminoacid at position 360; Ala for the amino acid at position 376; Ala forthe amino acid at position 380; Ala for the amino acid at position 382;Ala for the amino acid at position 384; Asp or His for the amino acid atposition 385; Pro for the amino acid at position 386; Glu for the aminoacid at position 387; Ala or Ser for the amino acid at position 389; Alafor the amino acid at position 424; Ala, Asp, Phe, Gly, His, Ile, Lys,Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr for the amino acid atposition 428; Lys for the amino acid at position 433; Ala, Phe, His,Ser, Trp, or Tyr for the amino acid at position 434; and His, Ile, Leu,Phe, Thr, or Val for the amino acid at position 436 as indicated by EUnumbering, in the amino acid sequence of the Fc region of SEQ ID NO: 5,6, 7, or
 8. 37. The antigen-binding molecule of any one of claims 29 and31 to 36, wherein the Fc region has a higher binding activity to aninhibitory Fcγ receptor than to an activating Fcγ receptor.
 38. Theantigen-binding molecule of claim 37, wherein the inhibitory Fcγreceptor is human FcγRIIb.
 39. The antigen-binding molecule of claim 37or 38, wherein the activating Fcγ receptor is human FcγRIa, humanFcγRIIa (R), human FcγRIIa (H), human FcγRIIIa (V), or human FcγRIIIa(F).
 40. The antigen-binding molecule of any one of claims 37 to 39,wherein the amino acid at position 238 or 328 (EU numbering) of the Fcregion includes an amino acid that is different from the amino acid ofthe native human IgG Fc region.
 41. The antigen-binding molecule ofclaim 40, wherein the amino acid at position 238 indicated by EUnumbering in the Fc region is Asp or the amino acid at position 328 isGlu.
 42. The antigen-binding molecule of claim 40 or 41, which comprisesat least one or more amino acids selected from the group consisting of:Asp for the amino acid at position 233; Trp or Tyr for the amino acid atposition 234; Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr for the aminoacid at position 237; Asp for the amino acid at position 239; Ala, Gln,or Val for the amino acid at position 267; Asn, Asp, or Glu for theamino acid at position 268; Gly for the amino acid at position 271; Ala,Asn, Asp, Gln, Glu, Leu, Met, Ser, or Thr for the amino acid at position326; Arg, Lys, or Met for the amino acid at position 330; Ile, Leu, orMet for the amino acid at position 323; and Asp for the amino acid atposition 296 according to EU numbering, in the amino acid sequence ofthe Fc region.
 43. The antigen-binding molecule of any one of claims 27to 42, wherein the antigen-binding molecule is an antibody.
 44. A methodfor producing the antigen-binding molecule of any one of claims 1 to 43,which comprises selecting an antigen-binding domain whoseantigen-binding activity varies depending on the concentration of atarget tissue-specific compound.
 45. A method of screening for theantigen-binding molecule of any one of claims 1 to 43, which comprisesselecting an antigen-binding domain whose antigen-binding activityvaries depending on the concentration of a target tissue-specificcompound.
 46. A pharmaceutical composition comprising theantigen-binding molecule of any one of claims 1 to 43.